Genes and uses for plant improvement

ABSTRACT

This invention provides transgenic plant cells with recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. This invention also provides transgenic plants and progeny seed comprising the transgenic plant cells where the plants are selected for having an enhanced trait selected from the group of traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Also disclosed are methods for manufacturing transgenic seed and plants with enhanced traits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 13/999,188, filed Jan. 23, 2014, which applicationis a continuation of and claims priority to U.S. patent application Ser.No. 11/330,903, filed Jan. 12, 2006, which claims the benefit ofpriority to U.S. provisional application Ser. No. 60/643,717, filed Jan.12, 2005, which applications are incorporated herein by reference andmade a part hereof in their entirety, and the benefit of priority ofeach of which is claimed herein.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computerreadable form (CRF) of the sequence listing, all on CD-ROMs, eachcontaining the file named “3126.003US3 seq list text(1354284x7ADA8).TXT”, which is 59,240,448 bytes (measured in MS-WINDOWS)and was created on Feb. 24, 2017, are herein incorporated by reference.

INCORPORATION OF COMPUTER PROGRAM LISTING

Computer Program Listing folders hmmer-2.3.2 and 158pfamDir arecontained on a compact disc and is hereby incorporated herein byreference in their entirety. Folder hmmer-2.3.2 contains the source codeand other associated file for implementing the HMMer software for Pfamanalysis. Folder 158pfamDir contains 158 Pfam Hidden Markov Models. Bothfolders were created on the disk on Feb. 28, 2017, having a total sizeof 15,616,000 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics anddevelopmental biology. More specifically, the present inventions providetransgenic seeds for crops, wherein the genome of said seed comprisesrecombinant DNA, the expression of which results in the production oftransgenic plants that have improved trait(s).

BACKGROUND OF THE INVENTION

Transgenic plants with improved traits such as improved yield,environmental stress tolerance, pest resistance, herbicide tolerance,modified seed compositions, and the like are desired by both farmers andconsumers. Although considerable efforts in plant breeding have providedsignificant gains in desired traits, the ability to introduce specificDNA into plant genomes provides further opportunities for generation ofplants with improved and/or unique traits. The ability to developtransgenic plants with improved traits depends in part on theidentification of genes that are useful in recombinant DNA constructsfor production of transformed plants with improved properties.

SUMMARY OF THE INVENTION (NEW)

This invention provides recombinant DNA for expression of proteins thatimpart enhanced agronomic traits in transgenic plants. Recombinant DNAin this invention is provided in a construct comprising a promoter thatis functional in plant cells and that is operably linked to DNA thatencodes a protein having at least one amino acid domain in a sequencethat exceeds the Pfam gathering cutoff for amino acid sequence alignmentwith a protein domain family identified by a Pfam name in the group ofPfam domain names identified in Table 17. In more specific embodimentsof the invention plant cells are provided which express a protein havingamino acid sequence with at least 90% identity to a consensus amino acidsequence in the group of consensus amino acid sequences consisting ofthe consensus amino acid sequence constructed for SEQ ID NO: 205 andhomologs thereof listed in Table 2 through the consensus amino acidsequence constructed for SEQ ID NO:408 and homologs thereof listed inTable 2. Amino acid sequences of homologs are SEQ ID NO:409 through19247. In even more specific embodiments of the invention the proteinexpressed in plant cells is a protein selected from the group ofproteins identified in Table 1 by annotation to a related protein inGenbank and alternatively identified in Table 16 by identification ofprotein domain family. An exemplary plant cell of this invention hasrecombinant DNA that encodes a protein identified by the Pdam name“RNA_pol_L”.

Other aspects of the invention are specifically directed to transgenicplant cells comprising the recombinant DNA of the invention, transgenicplants comprising a plurality of such plant cells, progeny transgenicseed, embryo and transgenic pollen from such plants. Such plant cellsare selected from a population of transgenic plants regenerated fromplant cells transformed with recombinant DNA and that express theprotein by screening transgenic plants in the population for an enhancedtrait as compared to control plants that do not have said recombinantDNA, where the enhanced trait is enhanced water use efficiency, enhancedcold tolerance, enhanced heat tolerance, enhanced shade tolerance,enhanced tolerance to salt exposure, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil. Thus, thisinvention provides transgenic plants and seeds with cells havingrecombinant DNA that impart at least one of those enhanced traits to theplants or seeds.

In yet another aspect of the invention the plant cells, plants, seeds,embryo and pollen further comprise DNA expressing a protein thatprovides tolerance from exposure to an herbicide applied at levels thatare lethal to a wild type of said plant cell. Such tolerance isespecially useful not only as an advantageous trait in such plants butis also useful in a selection step in the methods of the invention. Inaspects of the invention the agent of such herbicide is a glyphosate,dicamba, or glufosinate compound.

Yet other aspects of the invention provide transgenic plants which arehomozygous for the recombinant DNA and transgenic seed of the inventionfrom corn, soybean, cotton, canola, alfalfa, wheat or rice plants. Inother important embodiments for practice of various aspects of theinvention in Argentina the recombinant DNA is provided in plant cellsderived from corn lines that that are and maintain resistance to the Malde Rio Cuarto virus or the Puccina sorghi fungus or both.

This invention also provides methods for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith an enhanced trait resulting from expression of stably-integrated,recombinant DNA for expressing a protein having at least one domain ofamino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 17. Morespecifically the method comprises (a) screening a population of plantsfor an enhanced trait and recombinant DNA, where individual plants inthe population can exhibit the trait at a level less than, essentiallythe same as or greater than the level that the trait is exhibited incontrol plants which do not express the recombinant DNA, (b) selectingfrom the population one or more plants that exhibit the trait at a levelgreater than the level that said trait is exhibited in control plants,(c) verifying that the recombinant DNA is stably integrated in saidselected plants, (d) analyzing tissue of a selected plant to determinethe production of a protein having the function of a protein encoded bynucleotides in a sequence of one of SEQ ID NO:1-204; and (e) collectingseed from a selected plant. In one aspect of the invention the plants inthe population further comprise DNA expressing a protein that providestolerance to exposure to an herbicide applied at levels that are lethalto wild type plant cells and the selecting is effected by treating thepopulation with the herbicide, e.g. a glyphosate, dicamba, orglufosinate compound. In another aspect of the invention the plants areselected by identifying plants with the enhanced trait. The methods areespecially useful for manufacturing corn, soybean, cotton, alfalfa,wheat or rice seed selected as having one of the enhanced traitsdescribed above.

Another aspect of the invention provides a method of producing hybridcorn seed comprising acquiring hybrid corn seed from a herbicidetolerant corn plant which also has stably-integrated, recombinant DNAcomprising a promoter that is (a) functional in plant cells and (b) isoperably linked to DNA that encodes a protein having at least one domainof amino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 17. Themethods further comprise producing corn plants from said hybrid cornseed, where a fraction of the plants produced from said hybrid corn seedis homozygous for said recombinant DNA, a fraction of the plantsproduced from said hybrid corn seed is hemizygous for said recombinantDNA, and a fraction of the plants produced from said hybrid corn seedhas none of said recombinant DNA; selecting corn plants which arehomozygous and hemizygous for said recombinant DNA by treating with anherbicide; collecting seed from herbicide-treated-surviving corn plantsand planting said seed to produce further progeny corn plants; repeatingthe selecting and collecting steps at least once to produce an inbredcorn line; and crossing the inbred corn line with a second corn line toproduce hybrid seed.

Another aspect of the invention provides a method of selecting a plantcomprising plant cells of the invention by using an immunoreactiveantibody to detect the presence of protein expressed by recombinant DNAin seed or plant tissue. Yet another aspect of the invention providesanti-counterfeit milled seed having, as an indication of origin, a plantcells of this invention with unique recombinant DNA.

Another aspect of the invention provides plant cells having recombinantDNA for suppressing the expression of DNA identified in Table 1 andTable 16. More specific aspects of the invention provide plant tellshaving recombinant DNA for suppressing the expression of a proteinhaving the function in a plant of the protein with amino acid sequenceof SEQ ID NO: 213, 215, 218, 222, 258, 269, 275, 334, 361, 368, and 407or the corresponding Pfam identified in Table 16, i.e. Catalase,Bromdomain, FTCD_N, MatE, DPBB_1, tRNA-synt_2b, Sugar_tr and MFS_1, DUF6and DUF250, LEA_4, MIP and DUF231, respectively. Such suppression can beeffected by any of a number of ways known in the art, e.g. anti-sensesuppression, sense co-suppression, RNAi or knockout.

Still other specific aspects of this invention relate to growingtransgenic plants with enhanced water use efficiency or enhancednitrogen use efficiency. For instance, this invention provides methodsof growing a corn, cotton or soybean crop without irrigation watercomprising planting seed having plant cells of the invention which areselected for enhanced water use efficiency. Alternatively methodscomprise applying reduced irrigation water, e.g. providing up to 300millimeters of ground water during the production of a corn crop. Thisinvention also provides methods of growing a corn, cotton or soybeancrop without added nitrogen fertilizer comprising planting seed havingplant cells of the invention which are selected for enhanced nitrogenuse efficiency.

The various aspects of this invention are especially useful fortransgenic plant cells in seeds and transgenic plants having any of theabove-described enhanced traits in crop plants such as corn (maize),soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa,barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

The invention also comprises recombinant DNA constructs of the DNAuseful for imparting enhanced traits in plants having thee cells of thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show an alignment of amino acid sequences.

FIGS. 2 and 3 are plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO: 1-204 are DNA sequence of “genes” used in the recombinant DNAimparting an enhanced trait in plant cells;

SEQ ID NO:205-408 are amino acid sequence of the cognate protein ofthose “genes”;

SEQ ID NO:409-19247 are amino acid sequence of homologous proteins;

SEQ ID NO:19248 is a consensus amino acid sequence.

SEQ ID NO:19249 is a DNA sequence of a plasmid vector useful for corntransformation; and

SEQ ID NO:19250 is a DNA sequence of a plasmid vector useful for soybeantransformation.

As used herein, “gene” means DNA including chromosomal DNA, plasmid DNA,cDNA, synthetic DNA, or other DNA that is transcribed to RNA, e.g. mRNAthat encodes a protein or a protein fragment or anti-sense RNA or dsRNAfor suppression of expression of a target gene and its cognate protein.

“Transgenic plant cell” means a plant cell produced as an originaltransformation event, cells in plants regenerated from the originaltransformation, cells in progeny plants and seeds, and cells in plantsand seed from later generations or crosses of progeny plants and seeds,where such plant cells have recombinant DNA in their genome resultingfrom the original transformation.

“Recombinant DNA” means genetically engineered polynucleotide producedfrom endogenous and/or exogenous elements generally arranged as atranscription unit. Recombinant DNA may comprise DNA segments obtainedfrom different sources, or DNA segments obtained from the same source,but which have been manipulated to join DNA segments which do notnaturally exist in the joined form. A recombinant polynucleotide mayexist outside of the cell, for example as a PCR fragment, or integratedinto a genome, such as a plant genome.

“Trait” means to a physiological, morphological, biochemical, orphysical characteristic of a plant or particular plant material or cell.In some instances, this characteristic is visible to the human eye, suchas seed or plant size, or can be measured by biochemical techniques,such as detecting the protein, starch, or oil content of seed or leaves,or by observation of a metabolic or physiological process, e.g. bymeasuring uptake of carbon dioxide, or by the observation of theexpression level of a gene or genes, e.g., by employing Northernanalysis, RT-PCR, microarray gene expression assays, or reporter geneexpression systems, or by agricultural observations such as stresstolerance, yield, or pathogen tolerance. An “enhanced trait” as used indescribing the aspects of this invention includes enhanced water useefficiency, enhanced cold tolerance, enhanced heat tolerance, enhancedshade tolerance, enhanced tolerance to salt exposure, increased yield,enhanced nitrogen use efficiency, enhanced seed protein and enhancedseed oil.

As used herein, “control plant” is a plant without trait-improvingrecombinant DNA. A control plant is used to measure and compare traitimprovement in a transgenic plant with such trait-improving recombinantDNA. A suitable control plant may be a non-transgenic plant of theparental line used to generate a transgenic plant herein. Alternatively,a control plant may be a transgenic plant that comprises an empty vectoror marker gene, but does not contain the recombinant DNA that producesthe trait improvement. A control plant may also be a negative segregantprogeny of hemizygous transgenic plant. In certain demonstrations oftrait improvement, the use of a limited number of control plants cancause a wide variation in the control dataset. In analyzing trait dataduring screening to discover DNA useful in the plant cells of thisinvention it is useful to minimize the effect of the variation withinthe control dataset, i.e. a “reference” which is a trimmed mean of alldata from both transgenic and control plants grown under the sameconditions and at the same developmental stage. The trimmed mean iscalculated by eliminating a specific percentage, i.e. 20%, of thesmallest and largest observation from the data set and then calculatingthe average of the remaining observation. Many transgenic plantscomprising transgenic plant cells containing the recombinant DNAidentified herein as imparting an enhanced trait will not exhibit anenhanced agronomic trait. The transgenic plants and seeds comprising thetransgenic plant cells and having enhanced agronomic traits of thisinvention are identified by screening a population of transgenic plantsand/or seeds for the members of the population having the enhancedtrait. Screens for transgenic plant cells in crop plants are describedmore particularly in the examples below. In some cases, the traitenhancement can be measured quantitatively. For example, the traitenhancement can be at least a 2% desirable difference in an observedtrait, at least a 5% desirable difference, at least about a 10%desirable difference, at least about a 20% desirable difference, atleast about a 30% desirable difference, at least about a 50% desirabledifference, at least about a 70% desirable difference, or at least abouta 100% difference, or an even greater desirable difference. In othercases, the trait enhancement is measured qualitatively.

Many agronomic traits can affect “yield”, including without limitation,plant height, pod number, pod position on the plant, number ofinternodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Other traits that can affect yield include,efficiency of germination (including germination in stressedconditions), growth rate (including growth rate in stressed conditions),ear number, seed number per ear, seed size, composition of seed (starch,oil, protein) and characteristics of seed fill. Also of interest is thegeneration of transgenic plants that demonstrate desirable phenotypicproperties that may or may not confer an increase in overall plantyield. Such properties include enhanced plant morphology, plantphysiology or improved components of the mature seed harvested from thetransgenic plant.

“Stress condition” refers to the condition unfavorable for a plant,which adversely affect plant metabolism, growth and/or development. Aplant under the stress condition typically shows reduced germinationrate, retarded growth and development, reduced photosynthesis rate, andeventually leading to reduction in yield. Specifically, “water deficitstress” used herein preferably refers to the sub-optimal conditions forwater and humidity needed for normal growth of natural plants. Relativewater content (RWC) can be used as a physiological measure of plantwater deficit. It measures the effect of osmotic adjustment in plantwater status, when a plant is under stressed conditions. Conditionswhich may result in water deficit stress include heat, drought, highsalinity and PEG induced osmotic stress. “Cold stress” used hereinpreferably refers to the exposure of a plant to a temperatures below(two or more degrees Celsius below) those normal for a particularspecies or particular strain of plant. “Low nitrogen availabilitystress” used herein preferably refers to a plant growth condition with50% of the conventional nitrogen inputs. “Shade stress” used hereinpreferably refers to limited light availability that triggers the shadeavoidance response in plant. Plants are subject to shade stress whenlocalized at lower part of the canopy, or in close proximity ofneighboring vegetation. Shade stress may become exacerbated when theplanting density exceeds the average prevailing density for a particularplant species. The average prevailing densities per acre of a fewexamples of crop plants in the USA in the year 2000 were: wheat1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000,canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000plants per acre (Cheikh, et al., (2003) U.S. Patent Application No.20030101479).

“Increased yield” of a transgenic plant of the present invention may beevidenced and measured in a number of ways, including test weight, seednumber per plant, seed weight, seed number per unit area (i.e. seeds, orweight of seeds, per acre), bushels per acre, tons per acre, tons peracre, kilo per hectare. For example, maize yield may be measured asproduction of shelled corn kernels per unit of production area, e.g. inbushels per acre or metric tons per hectare, often reported on amoisture adjusted basis, e.g. at 15.5% moisture. Increased yield mayresult from improved utilization of key biochemical compounds, such asnitrogen, phosphorous and carbohydrate, or from improved tolerance toenvironmental stresses, such as cold, heat, drought, salt, and attack bypests or pathogens. Trait-enhancing recombinant DNA may also be used toprovide transgenic plants having improved growth and development, andultimately increased yield, as the result of modified expression ofplant growth regulators or modification of cell cycle or photosynthesispathways.

“Expression” means transcription of DNA to produce RNA. The resultingRNA may be without limitation mRNA encoding a protein, antisense RNAthat is complementary to an mRNA encoding a protein, or an RNAtranscript comprising a combination of sense and antisense gene regions,such as for use in RNAi technology. Expression as used herein may alsorefer to production of encoded protein from mRNA.

“Promoter” means a region of DNA that is upstream from the start oftranscription and is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissues are referred to as“tissue specific”. A “cell type” specific promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” or “repressible”promoter is a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions, or certain chemicals, or thepresence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterwhich is active under most conditions. As used herein, “antisenseorientation” includes reference to a polynucleotide sequence that isoperably linked to a promoter in an orientation where the antisensestrand is transcribed. The antisense strand is sufficientlycomplementary to an endogenous transcription product such thattranslation of the endogenous transcription product is often inhibited.“Operably linked” refers to the association of two or more nucleic acidfragments on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

“Consensus amino acid sequence” means an artificial, amino acid sequenceof conserved parts of the proteins encoded by homologous genes, e.g. asdetermined by a CLUSTALW alignment of amino acid sequence of homologproteins or a group of proteins having identified by the gatheringcutoff for a Pfam protein domain family.

Homologous genes are genes which encode homologous proteins with thesame or similar biological function or having the same Pfam proteindomain family. Homologous genes may be generated by the event ofspeciation (see ortholog) or by the event of genetic duplication (seeparalog). “Orthologs” refer to a set of homologous genes in differentspecies that evolved from a common ancestral gene by specification.Normally, orthologs retain the same function in the course of evolution;and “paralogs” refer to a set of homologous genes in the same speciesthat have diverged from each other as a consequence of geneticduplication. Thus, homologous genes can be from the same or a differentorganism. As used herein, “homolog” means a protein that performs thesame biological function as a second protein including those identifiedby sequence identity search.

“Percent identity” refers to the extent to which two optimally alignedDNA or protein segments are invariant throughout a window of alignmentof components, e.g. nucleotide sequence or amino acid sequence. An“identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by sequences of the two aligned segments divided by the totalnumber of sequence components in the reference segment over a window ofalignment which is the smaller of the full test sequence or the fullreference sequence. “Percent identity” (“% identity”) is the identityfraction times 100. “% identity to a consensus amino acid sequence” is100 times the identity fraction in a window of alignment of an aminoacid sequence of a test protein optimally aligned to consensus aminoacid sequence of this invention.

“Arabidopsis” means plants of Arabidopsis thaliana.

As used herein “Pfam” refers to a large collection of multiple sequencealignments and hidden Markov models covering many common proteinfamilies, e.g. Pfam version 18.0 (August 2005) contains alignments andmodels for 7973 protein families and is based on the Swissprot 47.0 andSP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “ProfileHidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam iscurrently maintained and updated by a Pfam Consortium. The alignmentsrepresent some evolutionary conserved structure that has implicationsfor the protein's function. Profile hidden Markov models (profile HMMs)built from the Pfam alignments are useful for automatically recognizingthat a new protein belongs to an existing protein family even if thehomology by alignment appears to be low. Once one DNA is identified asencoding a protein which imparts an enhanced trait when expressed intransgenic plants, other DNA encoding proteins in the same proteinfamily are identified by querying the amino acid sequence of proteinencoded by candidate DNA against the Hidden Markov Model whichcharacterizes the Pfam domain using HMMER software, a current version ofwhich is provided in the appended computer listing. Candidate proteinsmeeting the gathering cutoff for the alignment of a particular Pfam arein the protein family and have cognate DNA that is useful inconstructing recombinant DNA for the use in the plant cells of thisinvention. Hidden Markov Model databases for use with HMMER software inidentifying DNA expressing protein in a common Pfam for recombinant DNAin the plant cells of this invention are also included in the appendedcomputer listing. The HMMER software and Pfam databases are version 18.0and were used to identify known domains in the proteins corresponding toamino acid sequence of SEQ ID NO:205 through SEQ ID NO:408. All DNAencoding proteins that have scores higher than the gathering cutoffdisclosed in Table 27 by Pfam analysis disclosed herein can be used inrecombinant DNA of the plant cells of this invention, e.g. for selectingtransgenic plants having enhanced agronomic traits. The relevant Pfamsfor use in this invention, as more specifically disclosed below, are2-oxoacid_dh, ADH_N, ADH_zinc_N, AP2, AUX_IAA, Aa_trans, Abhydrolase_1,Acyl_transf_1, Aldedh, Aldo_ket_red, Alpha-amylase, Aminotran_1_2,Aminotran_3, Ammonium_transp, Arm, Asn_synthase, BAG, BSD,Beta_elim_lyase, Biotin_lipoyl, Brix, Bromodomain, C1_4, CTP_transf_2,Catalase, CcmH, Chal_sti_synt_C, Cyclin_C, Cyclin_N, Cys_Met_Meta_PP,DAO, DIM1, DPBB_1, DRMBL, DUF167, DUF231, DUF250, DUF6, DUF783, DUF962,E2F_TDP, E3_binding, EBP, Enolase_C, Enolase_N, F420_oxidored,FAD_binding_2, FA_desaturase, FKBP_C, FTCD_N, Fe_bilin_red, Fer4, GAF,GATase_2, GIDA, GSHPx, Gpi16, HGTP_anticodon, HI0933_like, HLH,HMG_CoA_synt, HWE_HK, Ham1p_like, HhH-GPD, Homeobox, Hpt, Iso_dh, K-box,LEA_4, LRRNT_2, LRR_1, Ldh_1_C, Ldh_1_N, Lectin_legA, Lectin_legB,Lipase_GDSL, MFS_1, MIP, MatE, Metalloenzyme, Methyltransf_11,Methyltransf_12, Molybdop_Fe4S4, Molybdopterin, Molydop_binding, Mov34,MtN3_slv, Myb_DNA-binding, NAD_Gly3P_dh_N, NAD_binding_2, NIR_SIR,NIR_SIR_ferr, NPH3, NTP_transferase, Nuc_sug_transp, PA, PAR1, PFK, PGI,PGK, PGM_PMM_I, PGM_PMM_II, PGM_PMM_III, PGM_PMM_IV, PP2C, PTR2,Peptidase_C26, Phi_1, Phytochrome, Pkinase, Pkinase_Tyr,Pollen_allerg_1, Pribosyltran, Proteasome, Pyr_redox, Pyr_redox_2,Pyr_redox_dim, RNA_pol_L, RNA_pol_Rpb6, RRM_1, RRN3, Radical_SAM, Ras,Response_reg, Rhodanese, Ribosomal_S8e, Rieske, SAC3_GANP, SBDS, SET,SRF-TF, SURF5, Skp1, Skp1_POZ, Ssl1, Sterol_desat, Sugar_tr, TCP, ThiF,Transaldolase, UQ_con, Ubie_methyltran, WD40, WRKY, adh_short, bZIP_1,bZIP_2, cNMP_binding, iPGM_N, p450, tRNA-synt_2 b, ubiquitin, zf-A20,zf-AN1, zf-B_box, zf-C2H2, zf-C3HC4, zf-CCCH, the databases for whichare included in the appended computer program listing.

Recombinant DNA Constructs

This invention provides recombinant DNA constructs comprising one ormore of the genes disclosed herein for imparting one or more enhancedtraits to transgenic plants and seeds. Such constructs also typicallycomprise a promoter operatively linked to said polynucleotide to providefor expression in a target plant. Other construct components may includeadditional regulatory elements, such as 5′ or 3′ untranslated regions(such as polyadenylation sites), intron regions, and transit or signalpeptides. Such recombinant DNA constructs can be assembled using methodsknown to those of ordinary skill in the art.

Recombinant constructs prepared in accordance with this inventiongenerally includes a 3′ untranslated DNA region (UTR) that typicallycontains a polyadenylation sequence following the polynucleotide codingregion. Examples of useful 3′ UTRs include those from the nopalinesynthase gene of Agrobacterium tumefaciens (nos), a gene encoding thesmall subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase(rbcS), and the T7 transcript of Agrobacterium tumefaciens and those 3′UTR elements disclosed in the following examples. Constructs and vectorsmay also include a transit peptide for targeting of a gene target to aplant organelle, particularly to a chloroplast, leucoplast or otherplastid organelle. For descriptions of the use of chloroplast transitpeptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925,incorporated herein by reference.

Table 1 provides a list of genes that can be used in recombinant DNA forimparting an enhanced trait in the transgenic plant cells, plants andseeds of this invention. In screens of recombinant DNA expressed in amodel plant the recombinant DNA was shown to be associated with enhancedtraits. The cognate protein was used to identify homologs forconstructing a consensus amino acid sequence for each cognate proteinand for identifying the characterizing Pfams. With reference to Table 1:

“NUC SEQ ID” refers to a SEQ ID NO. for particular DNA sequence in theSequence Listing;

“PEP SEQ ID” refers to a SEQ ID NO. in the Sequence Listing for theamino acid sequence of a protein cognate to a particular DNA;

“construct_id” refers to an arbitrary number used to identify aparticular recombinant DNA construct comprising the particular DNA;

“gene” refers to an arbitrary name used to identify the particular DNA;

“orientation” refers to the orientation of the particular DNA in arecombinant DNA construct relative to the promoter; and

“species name” refers to the organism from which the particular DNA wasderived.

TABLE 1 NUC PEP con- orien- SEQ ID SEQ ID struct_id Gene tation SpeciesName 1 205 12360 CGPG1018 SENSE Arabidopsis thaliana 2 206 12030CGPG1063 SENSE Arabidopsis thaliana 3 207 15210 CGPG1124 SENSEArabidopsis thaliana 4 208 17465 CGPG1178 SENSE Arabidopsis thaliana 5209 13222 CGPG1259 SENSE Arabidopsis thaliana 6 210 12919 CGPG1290 SENSEArabidopsis thaliana 7 211 12927 CGPG1300 SENSE Arabidopsis thaliana 8212 14841 CGPG1572 SENSE Arabidopsis thaliana 9 213 13478 CGPG1616 ANTI-Arabidopsis thaliana SENSE 10 214 17309 CGPG1840 SENSE Arabidopsisthaliana 11 215 19116 CGPG1861 ANTI- Arabidopsis thaliana SENSE 12 21614837 CGPG1882 SENSE Arabidopsis thaliana 13 217 71253 CGPG195 SENSEArabidopsis thaliana 14 218 16004 CGPG2061 ANTI- Arabidopsis thalianaSENSE 15 219 72712 CGPG2256 SENSE Arabidopsis thaliana 16 220 71546CGPG2305 SENSE Arabidopsis thaliana 17 221 70109 CGPG2368 SENSESaccharomyces cerevisiae 18 222 10335 CGPG246 ANTI- Arabidopsis thalianaSENSE 19 223 17919 CGPG2780 SENSE Arabidopsis thaliana 20 224 71148CGPG3225 SENSE Arabidopsis thaliana 21 225 19647 CGPG3229 SENSEArabidopsis thaliana 22 226 18844 CGPG3371 SENSE Arabidopsis thaliana 23227 19525 CGPG3548 SENSE Arabidopsis thaliana 24 228 19617 CGPG3573SENSE Arabidopsis thaliana 25 229 19750 CGPG3913 SENSE Saccharomycescerevisiae 26 230 19964 CGPG3922 SENSE Glycine max 27 231 19814 CGPG3934SENSE Glycine max 28 232 19720 CGPG3954 SENSE Glycine max 29 233 19845CGPG3956 SENSE Glycine max 30 234 71072 CGPG3963 SENSE Glycine max 31235 19949 CGPG4010 SENSE Glycine max 32 236 19902 CGPG4016 SENSE Glycinemax 33 237 19981 CGPG4019 SENSE Glycine max 34 238 19757 CGPG4037 SENSEGlycine max 35 239 19850 CGPG4056 SENSE Glycine max 36 240 19942CGPG4115 SENSE Glycine max 37 241 19787 CGPG4124 SENSE Glycine max 38242 19801 CGPG4137 SENSE Glycine max 39 243 19705 CGPG4156 SENSE Glycinemax 40 244 19737 CGPG4175 SENSE Glycine max 41 245 19812 CGPG4184 SENSEGlycine max 42 246 71556 CGPG4298 SENSE Arabidopsis thaliana 43 24771330 CGPG4457 SENSE Arabidopsis thaliana 44 248 71332 CGPG4460 SENSEArabidopsis thaliana 45 249 71571 CGPG4493 SENSE Arabidopsis thaliana 46250 70677 CGPG4558 SENSE Arabidopsis thaliana 47 251 71689 CGPG4661SENSE Glycine max 48 252 73685 CGPG4807 SENSE Arabidopsis thaliana 49253 72636 CGPG4858 SENSE Arabidopsis thaliana 50 254 73651 CGPG4869SENSE Arabidopsis thaliana 51 255 73342 CGPG4876 SENSE Arabidopsisthaliana 52 256 73271 CGPG5011 SENSE Arabidopsis thaliana 53 257 73282CGPG5071 SENSE Arabidopsis thaliana 54 258 10903 CGPG514 ANTI-Arabidopsis thaliana SENSE 55 259 73913 CGPG5337 SENSE Glycine max 56260 74305 CGPG5400 SENSE Arabidopsis thaliana 57 261 74306 CGPG5402SENSE Arabidopsis thaliana 58 262 73769 CGPG5430 SENSE Arabidopsisthaliana 59 263 74237 CGPG5448 SENSE Arabidopsis thaliana 60 264 74244CGPG5466 SENSE Arabidopsis thaliana 61 265 74256 CGPG5491 SENSEArabidopsis thaliana 62 266 72714 CGPG5513 SENSE Saccharomycescerevisiae 63 267 16014 CGPG552 SENSE Arabidopsis thaliana 64 268 72751CGPG5524 SENSE Saccharomyces cerevisiae 65 269 10814 CGPG554 ANTI-Arabidopsis thaliana SENSE 66 270 72743 CGPG5555 SENSE Saccharomycescerevisiae 67 271 72721 CGPG5569 SENSE Saccharomyces cerevisiae 68 27272959 CGPG5608 SENSE Glycine max 69 273 72984 CGPG5620 SENSE Arabidopsisthaliana 70 274 73061 CGPG5675 SENSE Rhodopseudomonas palustris 71 27510180 CGPG57 ANTI- Arabidopsis thaliana SENSE 72 276 73029 CGPG5737SENSE Saccharomyces cerevisiae 73 277 11145 CGPG574 SENSE Arabidopsisthaliana 74 278 72920 CGPG5763 SENSE Saccharomyces cerevisiae 75 27972957 CGPG5788 SENSE Saccharomyces cerevisiae 76 280 73044 CGPG5808SENSE Glycine max 77 281 74323 CGPG5873 SENSE Arabidopsis thaliana 78282 74327 CGPG5901 SENSE Arabidopsis thaliana 79 283 74329 CGPG5903SENSE Arabidopsis thaliana 80 284 74340 CGPG5907 SENSE Arabidopsisthaliana 81 285 74341 CGPG5911 SENSE Arabidopsis thaliana 82 286 74345CGPG5932 SENSE Arabidopsis thaliana 83 287 74616 CGPG6017 SENSEArabidopsis thaliana 84 288 74604 CGPG6019 SENSE Arabidopsis thaliana 85289 74608 CGPG6043 SENSE Arabidopsis thaliana 86 290 74609 CGPG6044SENSE Arabidopsis thaliana 87 291 74366 CGPG6073 SENSE Arabidopsisthaliana 88 292 74383 CGPG6098 SENSE Arabidopsis thaliana 89 293 74374CGPG6100 SENSE Arabidopsis thaliana 90 294 74618 CGPG6117 SENSEArabidopsis thaliana 91 295 74619 CGPG6118 SENSE Arabidopsis thaliana 92296 74622 CGPG6124 SENSE Arabidopsis thaliana 93 297 74628 CGPG6131SENSE Arabidopsis thaliana 94 298 74631 CGPG6139 SENSE Arabidopsisthaliana 95 299 74669 CGPG6140 SENSE Arabidopsis thaliana 96 300 74670CGPG6145 SENSE Arabidopsis thaliana 97 301 74647 CGPG6163 SENSEArabidopsis thaliana 98 302 74385 CGPG6310 SENSE Arabidopsis thaliana 99303 74386 CGPG6330 SENSE Arabidopsis thaliana 100 304 74387 CGPG6331SENSE Arabidopsis thaliana 101 305 74685 CGPG6362 SENSE Arabidopsisthaliana 102 306 73487 CGPG6386 SENSE Sinorhizobium meliloti 103 30773476 CGPG6393 SENSE Bacillus subtilis subsp. subtilis str. 168 104 30873465 CGPG6400 SENSE Escherichia coli K12 105 309 73418 CGPG6404 SENSENostoc punctiforme PCC 73102 106 310 73480 CGPG6425 SENSE Sinorhizobiummeliloti 107 311 73482 CGPG6438 SENSE Agrobacterium tumefaciens str. C58108 312 73513 CGPG6457 SENSE Bacillus subtilis subsp. subtilis str. 168109 313 73550 CGPG6468 SENSE Bacillus halodurans 110 314 73527 CGPG6474SENSE Desulfitobacterium hafniense 111 315 73516 CGPG6481 SENSEXenorhabdus nematophila 112 316 73530 CGPG6498 SENSE Escherichia coliK12 113 317 73534 CGPG6527 SENSE Bacillus subtilis subsp. subtilis str.168 114 318 74125 CGPG6544 SENSE Pseudomonas fluorescens PfO-1 115 31974161 CGPG6547 SENSE Escherichia coli K12 116 320 74126 CGPG6552 SENSEEscherichia coli K12 117 321 74115 CGPG6559 SENSE Escherichia coli K12118 322 74127 CGPG6560 SENSE Escherichia coli K12 119 323 74128 CGPG6568SENSE Escherichia coli K12 120 324 74165 CGPG6579 SENSE Escherichia coliK12 121 325 74106 CGPG6582 SENSE Pseudomonas fluorescens PfO-1 122 32674130 CGPG6584 SENSE Pseudomonas syringae pv. tomato str. DC3000 123 32774132 CGPG6600 SENSE Xenorhabdus nematophila 124 328 74144 CGPG6601SENSE Xenorhabdus nematophila 125 329 74402 CGPG6663 SENSE Synechocystissp. 126 330 74476 CGPG6685 SENSE Bacillus halodurans 127 331 74417CGPG6688 SENSE Bacillus halodurans 128 332 74453 CGPG6691 SENSE Bacillussubtilis subsp. subtilis str. 168 129 333 74418 CGPG6696 SENSEEscherichia coli K12 130 334 10150 CGPG67 ANTI- Arabidopsis thalianaSENSE 131 335 74503 CGPG6767 SENSE Escherichia coli K12 132 336 74504CGPG6775 SENSE Agrobacterium tumefaciens 133 337 74528 CGPG6777 SENSEBacillus halodurans 134 338 74588 CGPG6782 SENSE Escherichia coli K12135 339 74541 CGPG6786 SENSE Escherichia coli K12 136 340 74553 CGPG6787SENSE Nostoc punctiforme PCC 73102 137 341 74554 CGPG6795 SENSEPseudomonas syringae pv. tomato str. DC3000 138 342 74578 CGPG6797 SENSESynechocystis sp. PCC 6803 139 343 74590 CGPG6798 SENSE Xenorhabdusnematophila 140 344 75834 CGPG6820 SENSE Arabidopsis thaliana 141 34575835 CGPG6822 SENSE Arabidopsis thaliana 142 346 18020 CGPG684 SENSEArabidopsis thaliana 143 347 75850 CGPG6893 SENSE Arabidopsis thaliana144 348 75861 CGPG6937 SENSE Arabidopsis thaliana 145 349 75875 CGPG6992SENSE Arabidopsis thaliana 146 350 74548 CGPG712 SENSE Arabidopsisthaliana 147 351 74880 CGPG7357 SENSE Glycine max 148 352 74903 CGPG7407SENSE Zea mays 149 353 74915 CGPG7408 SENSE Zea mays 150 354 74927CGPG7409 SENSE Zea mays 151 355 74951 CGPG7411 SENSE Glycine max 152 35674940 CGPG7418 SENSE Zea mays 153 357 74977 CGPG7429 SENSE Synechocystissp. 154 358 74954 CGPG7435 SENSE Xanthomonas campestris 155 359 74907CGPG7439 SENSE Synechocystis sp. 156 360 74919 CGPG7440 SENSESynechocystis sp. 157 361 11735 CGPG745 ANTI- Arabidopsis thaliana SENSE158 362 74980 CGPG7453 SENSE Synechocystis sp. 159 363 74993 CGPG7462SENSE Bacillus halodurans 160 364 75337 CGPG7469 SENSE Saccharomycescerevisiae 161 365 75339 CGPG7485 SENSE Zea mays 162 366 75316 CGPG7491SENSE Glycine max 163 367 75352 CGPG7494 SENSE Zea mays 164 368 12189CGPG752 ANTI- Arabidopsis thaliana SENSE 165 369 75321 CGPG7531 SENSEZea mays 166 370 75358 CGPG7542 SENSE Zea mays 167 371 75312 CGPG7554SENSE Zea mays 168 372 75463 CGPG7583 SENSE Zea mays 169 373 75475CGPG7584 SENSE Zea mays 170 374 75440 CGPG7589 SENSE Glycine max 171 37575488 CGPG7593 SENSE Glycine max 172 376 75418 CGPG7603 SENSE Glycinemax 173 377 75419 CGPG7611 SENSE Zea mays 174 378 75431 CGPG7612 SENSEGlycine max 175 379 75455 CGPG7614 SENSE Glycine max 176 380 75491CGPG7617 SENSE Zea mays 177 381 75456 CGPG7622 SENSE Glycine max 178 38275480 CGPG7624 SENSE Glycine max 179 383 75492 CGPG7625 SENSE Zea mays180 384 75409 CGPG7626 SENSE Zea mays 181 385 75424 CGPG7651 SENSE Zeamays 182 386 75550 CGPG7676 SENSE Glycine max 183 387 75575 CGPG7686SENSE Glycine max 184 388 75528 CGPG7690 SENSE Glycine max 185 389 75564CGPG7693 SENSE Glycine max 186 390 75553 CGPG7700 SENSE Glycine max 187391 75506 CGPG7704 SENSE Glycine max 188 392 75554 CGPG7708 SENSEGlycine max 189 393 75590 CGPG7711 SENSE Glycine max 190 394 75543CGPG7715 SENSE Glycine max 191 395 75567 CGPG7717 SENSE Glycine max 192396 75544 CGPG7723 SENSE Glycine max 193 397 75556 CGPG7724 SENSEGlycine max 194 398 75546 CGPG7739 SENSE Glycine max 195 399 75558CGPG7740 SENSE Glycine max 196 400 75571 CGPG7749 SENSE Glycine max 197401 75583 CGPG7750 SENSE Glycine max 198 402 75536 CGPG7754 SENSE Zeamays 199 403 75991 CGPG8266 SENSE Chloroflexus aurantiacus 200 404 75909CGPG8275 SENSE Chloroflexus aurantiacus 201 405 12824 CGPG885 SENSEArabidopsis thaliana 202 406 18026 CGPG894 SENSE Arabidopsis thaliana203 407 11810 CGPG899 ANTI- Arabidopsis thaliana SENSE 204 408 72418CGPG968 SENSE Arabidopsis thaliana

Recombinant DNA

Exemplary DNA for use in the present invention to improve traits inplants are provided herein as SEQ ID NO:1 through SEQ ID NO:204, as wellas the homologs of such DNA molecules. A subset of the exemplary DNAincludes fragments of the disclosed full polynucleotides consisting ofoligonucleotides of at least 15, preferably at least 16 or 17, morepreferably at least 18 or 19, and even more preferably at least 20 ormore, consecutive nucleotides. Such oligonucleotides are fragments ofthe larger molecules having a sequence selected from the groupconsisting of SEQ ID NO:1 through SEQ ID NO:204, and find use, forexample as probes and primers for detection of the polynucleotides ofthe present invention.

Also of interest in the present invention are variants of the DNAprovided herein. Such variants may be naturally occurring, including DNAfrom homologous genes from the same or a different species, or may benon-natural variants, for example DNA synthesized using chemicalsynthesis methods, or generated using recombinant DNA techniques.Degeneracy of the genetic code provides the possibility to substitute atleast one base of the protein encoding sequence of a gene with adifferent base without causing the amino acid sequence of thepolypeptide produced from the gene to be changed. Hence, a DNA useful inthe present invention may have any base sequence that has been changedfrom the sequences provided herein by substitution in accordance withdegeneracy of the genetic code.

Homologs of the genes providing DNA of demonstrated as useful inimproving traits in model plants disclosed herein will generallydemonstrate significant identity with the DNA provided herein. DNA issubstantially identical to a reference DNA if, when the sequences of thepolynucleotides are optimally aligned there is about 60% nucleotideequivalence; more preferably 70%; more preferably 80% equivalence; morepreferably 85% equivalence; more preferably 90%; more preferably 95%;and/or more preferably 98% or 99% equivalence over a comparison window.A comparison window is preferably at least 50-100 nucleotides, and morepreferably is the entire length of the polynucleotide provided herein.Optimal alignment of sequences for aligning a comparison window may beconducted by algorithms; preferably by computerized implementations ofthese algorithms (for example, the Wisconsin Genetics Software PackageRelease 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.). The reference polynucleotide may be a full-length molecule or aportion of a longer molecule. Preferentially, the window of comparisonfor determining polynucleotide identity of protein encoding sequences isthe entire coding region.

Proteins useful for imparting improved traits are entire proteins or atleast a sufficient portion of the entire protein to impart the relevantbiological activity of the protein. The term “protein” also includesmolecules consisting of one or more polypeptide chains. Thus, a proteinuseful in the present invention may constitute an entire protein havingthe desired biological activity, or may constitute a portion of anoligomeric protein having multiple polypeptide chains. Proteins usefulfor generation of transgenic plants having improved traits include theproteins with an amino acid sequence provided herein as SEQ ID NO: 205through SEQ ID NO: 408, as well as homologs of such proteins.

Homologs of the proteins useful in the present invention may beidentified by comparison of the amino acid sequence of the protein toamino acid sequences of proteins from the same or different plantsources, e.g. manually or by using known homology-based searchalgorithms such as those commonly known and referred to as BLAST, FASTA,and Smith-Waterman. As used herein, a homolog is a protein from the sameor a different organism that performs the same biological function asthe polypeptide to which it is compared. An orthologous relation betweentwo organisms is not necessarily manifest as a one-to-one correspondencebetween two genes, because a gene can be duplicated or deleted afterorganism phylogenetic separation, such as speciation. For a givenprotein, there may be no ortholog or more than one ortholog. Othercomplicating factors include alternatively spliced transcripts from thesame gene, limited gene identification, redundant copies of the samegene with different sequence lengths or corrected sequence. A localsequence alignment program, e.g. BLAST, can be used to search a databaseof sequences to find similar sequences, and the summary Expectationvalue (E-value) used to measure the sequence base similarity. As aprotein hit with the best E-value for a particular organism may notnecessarily be an ortholog or the only ortholog, a reciprocal BLASTsearch is used in the present invention to filter hit sequences withsignificant E-values for ortholog identification. The reciprocal BLASTentails search of the significant hits against a database of amino acidsequences from the base organism that are similar to the sequence of thequery protein. A hit is a likely ortholog, when the reciprocal BLAST'sbest hit is the query protein itself or a protein encoded by aduplicated gene after speciation. Thus, homolog is used herein todescribed proteins that are assumed to have functional similarity byinference from sequence base similarity. The relationship of homologswith amino acid sequences of SEQ ID NO:409 to 19247 to the proteins withamino acid sequences of SEQ ID NO:206 to 408 is found in the listing ofTable 2.

A further aspect of the invention comprises functional homolog proteinswhich differ in one or more amino acids from those of a trait-improvingprotein disclosed herein as the result of one or more of the well-knownconservative amino acid substitutions, e.g. valine is a conservativesubstitute for alanine and threonine is a conservative substitute forserine. Conservative substitutions for an amino acid within the nativesequence can be selected from other members of a class to which thenaturally occurring amino acid belongs. Representative amino acidswithin these various classes include, but are not limited to: (1) acidic(negatively charged) amino acids such as aspartic acid and glutamicacid; (2) basic (positively charged) amino acids such as arginine,histidine, and lysine; (3) neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and(4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.Conserved substitutes for an amino acid within a native amino acidsequence can be selected from other members of the group to which thenaturally occurring amino acid belongs. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Naturallyconservative amino acids substitution groups are: valine-leucine,valine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Afurther aspect of the invention comprises proteins that differ in one ormore amino acids from those of a described protein sequence as theresult of deletion or insertion of one or more amino acids in a nativesequence.

Homologs of the trait-improving proteins disclosed provided herein willgenerally demonstrate significant sequence identity. Of particularinterest are proteins having at least 50% sequence identity, morepreferably at least about 70% sequence identity or higher, e.g. at leastabout 80% sequence identity with an amino acid sequence of SEQ ID NO:205 through SEQ ID NO: 408. Of course useful proteins also include thosewith higher identity, e.g. 90% to 99% identity. Identity of proteinhomologs is determined by optimally aligning the amino acid sequence ofa putative protein homolog with a defined amino acid sequence and bycalculating the percentage of identical and conservatively substitutedamino acids over the window of comparison. The window of comparison fordetermining identity can be the entire amino acid sequence disclosedherein, e.g. the full sequence of any of SEQ ID NO:205 through SEQ IDNO:408.

Genes that are homologous to each other can be grouped into families andincluded in multiple sequence alignments. Then a consensus sequence foreach group can be derived. This analysis enables the derivation ofconserved and class- (family) specific residues or motifs that arefunctionally important. These conserved residues and motifs can befurther validated with 3D protein structure if available. The consensussequence can be used to define the full scope of the invention, e.g. toidentify proteins with a homolog relationship. Thus, the presentinvention contemplates that protein homologs include proteins with anamino acid sequence that has at least 90% identity to such a consensusamino acid sequence sequences.

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the proteinsdisclosed herein. Means for preparing and characterizing antibodies arewell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified antifungal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. The antibody-producing B lymphocytesfrom the immunized animal are then fused with cells of an immortalmyeloma cell, generally one of the same species as the animal that wasimmunized to establish a population of hybridomas from which specifichybridomas are selected. The selected hybridomas would then be seriallydiluted and cloned into individual antibody-producing cell lines, whichclones can then be propagated indefinitely to provide mAbs.

Promoters

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which disclosesa maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a riceactin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter,U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat.No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat.No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No.6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat.No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Patent Application Publication 2002/0192813A1 which discloses 5′,3′ and intron elements useful in the design of effective plantexpression vectors, U.S. patent application Ser. No. 09/078,972 whichdiscloses a coixin promoter, U.S. patent application Ser. No. 09/757,089which discloses a maize chloroplast aldolase promoter, and U.S. patentapplication Ser. No. 10/739,565 which discloses water-deficit induciblepromoters, all of which are incorporated herein by reference. These andnumerous other promoters that function in plant cells are known to thoseskilled in the art and available for use in recombinant polynucleotidesof the present invention to provide for expression of desired genes intransgenic plant cells.

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedin the forward or reverse orientation 5′ or 3′ to the coding sequence.In some instances, these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1and rice actin 2 genes. Examples of other enhancers that can be used inaccordance with the invention include elements from the CaMV 35Spromoter, octopine synthase genes, the maize alcohol dehydrogenase gene,the maize shrunken 1 gene and promoters from non-plant eukaryotes.

In some aspects of the invention it is preferred that the promoterelement in the DNA construct be capable of causing sufficient expressionto result in the production of an effective amount of a polypeptide inwater deficit conditions. Such promoters can be identified and isolatedfrom the regulatory region of plant genes that are over expressed inwater deficit conditions. Specific water-deficit-inducible promoters foruse in this invention are derived from the 5′ regulatory region of genesidentified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene(HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene(CA4H) of Zea maize. Such water-deficit-inducible promoters aredisclosed in U.S. application Ser. No. 10/739,565, incorporated hereinby reference.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216).

In still other aspects of the invention, preferential expression inplant green tissues is desired. Promoters of interest for such usesinclude those from genes such as SSU (Fischhoff et al. (1992) Plant MolBiol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

Gene Overexpression

“Gene overexpression” used herein in reference to a polynucleotide orpolypeptide indicates that the expression level of a target protein, ina transgenic plant or in a host cell of the transgenic plant, exceedslevels of expression in a non-transgenic plant. In a preferredembodiment of the present invention, a recombinant DNA constructcomprises the polynucleotide of interest in the sense orientationrelative to the promoter to achieve gene overexpression, which isidentified as such in Table 1.

Gene Suppression

Gene suppression includes any of the well-known methods for suppressingtranscription of a gene or the accumulation of the mRNA corresponding tothat gene thereby preventing translation of the transcript into protein.Posttranscriptional gene suppression is mediated by transcription ofintegrated recombinant DNA to form double-stranded RNA (dsRNA) havinghomology to a gene targeted for suppression. This formation of dsRNAmost commonly results from transcription of an integrated invertedrepeat of the target gene, and is a common feature of gene suppressionmethods known as anti-sense suppression, co-suppression, RNAinterference (RNAi) and knockout, e.g. by mutagenesis. Transcriptionalsuppression can be mediated by a transcribed dsRNA having homology to apromoter DNA sequence to effect what is called promoter transsuppression.

More particularly, posttranscriptional gene suppression by inserting arecombinant DNA construct with anti-sense oriented DNA to regulate geneexpression in plant cells is disclosed in U.S. Pat. No. 5,107,065(Shewmaker et al.) and U.S. Pat. No. 5,759,829 (Shewmaker et al.).Transgenic plants transformed using such anti-sense oriented DNAconstructs for gene suppression can comprise integrated DNA arranged asan inverted repeats that result from insertion of the DNA construct intoplants by Agrobacterium-mediated transformation, as disclosed byRedenbaugh et al. in “Safety Assessment of Genetically Engineered FlavrSavr™ Tomato, CRC Press, Inc. (1992). Inverted repeat insertions cancomprises a part or all of the T-DNA construct, e.g. an inverted repeatof a complete transcription unit or an inverted repeat of transcriptionterminator sequence. Screening for inserted DNA comprising invertedrepeat elements can improve the efficiency of identifying transformationevents effective for gene silencing whether the transformation constructis a simple anti-sense DNA construct which must be inserted in multiplecopies or a complex inverted repeat DNA construct (e.g. an RNAiconstruct) which can be inserted as a single copy.

Posttranscriptional gene suppression by inserting a recombinant DNAconstruct with sense-oriented DNA to regulate gene expression in plantsis disclosed in U.S. Pat. No. 5,283,184 (Jorgensen et al.) and U.S. Pat.No. 5,231,020 (Jorgensen et al.). Inserted T-DNA providing genesuppression in plants transformed with such sense constructs byAgrobacterium is organized predominately in inverted repeat structures,as disclosed by Jorgensen et al., Mol. Gen. Genet., 207:471-477 (1987).See also Stam et al., The Plant Journal, 12(1), 63-82 (1997) who usedsegregation studies to support Jorgensen's finding that gene silencingis mediated by multimeric transgene T-DNA loci in which the T-DNAs arearranged in inverted repeats. Screening for inserted DNA comprisinginverted repeat elements can improve the gene silencing efficiency whentransforming with simple sense-orientated DNA constructs. Gene silencingefficiency can also be improved by screening for single insertion eventswhen transforming with an RNAi construct containing inverted repeatelements

As disclosed by Redenbaugh et al. gene suppression can be achieved byinserting into a plant genome recombinant DNA that transcribes dsRNA.Such a DNA insert can be transcribed to an RNA element having the 3′region as a double stranded RNA. RNAi constructs are also disclosed inEP 0426195 A1 (Goldbach et al.—1991) where recombinant DNA constructsfor transcription into hairpin dsRNA for providing transgenic plantswith resistance to tobacco spotted wilt virus. Double-stranded RNAs werealso disclosed in WO 94/01550 (Agrawal et al.) where anti-sense RNA wasstabilized with a self-complementary 3′ segment. Agrawal et al. referredto U.S. Pat. No. 5,107,065 for using such self-stablized anti-sense RNAsfor regulating gene expression in plant cells; see InternationalPublication No. 94/01550. Other double-stranded hairpin-forming elementsin transcribed RNA are disclosed in International Publication No.98/05770 (Werner et al.) where the anti-sense RNA is stabilized byhairpin forming repeats of poly(CG) nucleotides. See also U.S. PatentApplication Publication No. 2003/0175965 A 1 (Lowe et al.) whichdiscloses gene suppression using and RNAi construct comprising a genecoding sequence preceded by inverted repeats of 5′UTR. See also U.S.Patent Application Publication No. 2002/0048814 A1 (Oeller) where RNAiconstructs are transcribed to sense or anti-sense RNA which isstabilized by a poly(T)-poly(A) tail. See also U.S. Patent ApplicationPublication No. 2003/0018993 A1 (Gutterson et al.) where sense oranti-sense RNA is stabilized by an inverted repeat of a of the 3′untranslated region of the NOS gene. See also U.S. Patent ApplicationPublication No. 2003/0036197 A1 (Glassman et al.) where RNA havinghomology to a target is stabilized by two complementary RNA regions.

Gene silencing can also be effected by transcribing RNA from both asense and an anti-sense oriented DNA, e.g. as disclosed by Shewmaker etal. in U.S. Pat. No. 5,107,065 where in Example 1 a binary vector wasprepared with both sense and anti-sense aroA genes. See also U.S. Pat.No. 6,326,193 where gene targeted DNA is operably linked to opposingpromoters.

Gene silencing can also be affected by transcribing from contiguoussense and anti-sense DNA. In this regard see Sijen et al., The PlantCell, Vol. 8, 2277-2294 (1996) discloses the use of constructs carryinginverted repeats of a cowpea mosaic virus gene in transgenic plants tomediate virus resistance. Such constructs for posttranscriptional genesuppression in plants by double-stranded RNA are also disclosed inInternational Publication No. WO 99/53050 (Waterhouse et al.),International Publication No. WO 99/49029 (Graham et al.), U.S. patentapplication Ser. No. 10/465,800 (Fillatti), U.S. Pat. No. 6,506,559(Fire et al.). See also U.S. application Ser. No. 10/393,347 (Shewmakeret al.) that discloses constructs and methods for simultaneouslyexpressing one or more recombinant genes while simultaneouslysuppressing one or more native genes in a transgenic plant. See alsoU.S. Pat. No. 6,448,473 (Mitsky et al.) that discloses multi-genesuppression vectors for use in plants. All of the above-describedpatents, applications and international publications disclosingmaterials and methods for posttranscriptional gene suppression in plantsare incorporated herein by reference. Transcriptional suppression suchas promoter trans suppression can be affected by a expressing a DNAconstruct comprising a promoter operably linked to inverted repeats ofpromoter DNA for a target gene. Constructs useful for such genesuppression mediated by promoter trans suppression are disclosed byMette et al., The EMBO Journal, Vol. 18, No. 1, pp. 241-148, 1999 and byMette et al., The EMBO Journal, Vol. 19, No. 19, pp. 5194-5201-148,2000, both of which are incorporated herein by reference.

Suppression can also be achieved by insertion mutations created bytransposable elements may also prevent gene function. For example, inmany dicot plants, transformation with the T-DNA of Agrobacterium may bereadily achieved and large numbers of transformants can be rapidlyobtained. Also, some species have lines with active transposableelements that can efficiently be used for the generation of largenumbers of insertion mutations, while some other species lack suchoptions. Mutant plants produced by Agrobacterium or transposonmutagenesis and having altered expression of a polypeptide of interestcan be identified using the polynucleotides of the present invention.For example, a large population of mutated plants may be screened withpolynucleotides encoding the polypeptide of interest to detect mutatedplants having an insertion in the gene encoding the polypeptide ofinterest.

Gene Stacking

The present invention also contemplates that the trait-improvingrecombinant DNA provided herein can be used in combination with otherrecombinant DNA to create plants with a multiple desired traits. Thecombinations generated can include multiple copies of any one or more ofthe recombinant DNA constructs. These stacked combinations can becreated by any method, including but not limited to cross breeding oftransgenic plants, or multiple genetic transformation.

Plant Cell Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, for example various mediaand recipient target cells, transformation of immature embryo cells andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for selection of plantshaving an enhanced trait. In addition to direct transformation of aplant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant which can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced trait, e.g. enhancedyield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, for example herbicideresistance or pest resistance, to produce progeny plants havingrecombinant DNA that confers both traits. Typically, in such breedingfor combining traits the transgenic plant donating the additional traitis a male line and the transgenic plant carrying the base traits is thefemale line. The progeny of this cross will segregate such that some ofthe plants will carry the DNA for both parental traits and some willcarry DNA for one parental trait; such plants can be identified bymarkers associated with parental recombinant DNA, e.g. markeridentification by analysis for recombinant DNA or, in the case where aselectable marker is linked to the recombinant, by application of theselecting agent such as a herbicide for use with a herbicide tolerancemarker, or by selection for the enhanced trait. Progeny plants carryingDNA for both parental traits can be crossed back into the female parentline multiple times, for example usually 6 to 8 generations, to producea progeny plant with substantially the same genotype as one originaltransgenic parental line but for the recombinant DNA of the othertransgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which plants of this invention may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of whichare incorporated herein by reference. Selectable markers which providean ability to visually identify transformants can also be employed, forexample, a gene expressing a colored or fluorescent protein such as aluciferase or green fluorescent protein (GFP) or a gene expressing abeta-glucuronidase or uidA gene (GUS) for which various chromogenicsubstrates are known.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to agreenhouse or growth chamber for maturation. Plants are regenerated fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced,for example self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells of this invention aregrown to generate transgenic plants having an enhanced trait as comparedto a control plant and produce transgenic seed and haploid pollen ofthis invention. Such plants with enhanced traits are identified byselection of transformed plants or progeny seed for the enhanced trait.For efficiency a selection method is designed to evaluate multipletransgenic plants (events) comprising the recombinant DNA, for examplemultiple plants from 2 to 20 or more transgenic events. Transgenicplants grown from transgenic seed provided herein demonstrate improvedagronomic traits that contribute to increased yield or other trait thatprovides increased plant value, including, for example, improved seedquality. Of particular interest are plants having enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil.

Discovery of Trait-improving Recombinant DNA

To identify recombinant DNA that imparts an enhanced trait to plants,Arabidopsis cells were transformed with a candidate recombinant DNAconstruct and screened for an improved trait. A two-step screeningprocess was employed which comprised two passes of traitcharacterization to ensure that the trait modification was dependent onexpression of the recombinant DNA, but not due to the chromosomallocation of the integration of the transgene. Twelve independenttransgenic lines for each recombinant DNA construct were established andassayed for the transgene expression levels. Five transgenic lines withhigh transgene expression is levels were used in the first pass screento evaluate the transgene's function in T2 transgenic plants.Subsequently, three transgenic events, which had been shown to have oneor more improved traits, were further evaluated in the second passscreen to confirm the transgene's ability to impart an improved trait.The following Table 3 summarizes the improved traits that have beenconfirmed as provided by a recombinant DNA construct.

In particular, Table 3 reports

“PEP SEQ ID” which is the amino acid sequence of the protein cognate tothe DNA in the recombinant DNA construct corresponding to a proteinsequence of a SEQ ID NO. in the Sequence Listing.“annotation” refers to a description of the top hit protein obtainedfrom an amino acid sequence query of each PEP SEQ ID NO to GenBankdatabase of the National Center for Biotechnology Information (ncbi).More particularly, “gi” is the GenBank ID number for the top BLAST hit.The components of “annotation” are “e-value” which provides theexpectation value for the BLAST hit; “% id” which refers to thepercentage of identically matched amino acid residues along the lengthof the portion of the sequences which is aligned by BLAST between thesequence of interest provided herein and the hit sequence in GenBank;“GenBank ID” which provides a reference number for the top BLAST hit inGenBank; and “description” which refers to the description of that topBLAST hit.“traits” identify by two letter codes the confirmed improvement in atransgenic plant provided by the recombinant DNA. The codes for improvedtraits are:“CK” which indicates cold tolerance improvement identified under a coldshock tolerance screen;“CS” which indicates cold tolerance improvement identified by a coldgermination tolerance screen;“DS” which indicates drought tolerance improvement identified by a soildrought stress tolerance screen;“PEG” which indicates osmotic stress tolerance improvement identified bya PEG induced osmotic stress tolerance screen;“HS” which indicates heat stress tolerance improvement identified by aheat stress tolerance screen;“SS” which indicates high salinity stress tolerance improvementidentified by a salt stress tolerance screen;“LN” which indicates nitrogen use efficiency improvement identified by alimited nitrogen tolerance screen;“LL” which indicates attenuated shade avoidance response identified by ashade tolerance screen under a low light condition;“PP” which indicates improved growth and development at early stagesidentified by an early plant growth and development screen;“SP” which indicates improved growth and development at late stagesidentified by a late plant growth and development screen providedherein.

TABLE 3 PEP annotation SEQ e- % ID value id GenBank ID descriptionTraits 205 5.00E−34 85 gi|21281159| gb|AAD48981.1|contains HS similarityto Solanum lycopersicum (tomato) wound induced protein 206 1.00E−66 76gi|21553397| gb|AAM62490.1|putative zinc LN finger protein 207 1.00E−11893 gi|7573441| ref|NP_191871.1|cyclin HS family protein 208 0 93gi|22136838| ref|NP_566299.1|GPI CK CS transamidase component Gpi16subunit family protein 209 1.00E−171 97 gi|15028251| ref|NP_566244.1| CKtransmembrane protein, putative 210 2.00E−57 98 gi|37202020|emb|CAB81279.1|putative HS PP PEG protein 211 8.00E−37 100 gi|26451972|dbj|BAC43077.1|unknown PP SS HS protein 212 0 86 gi|42562765|ref|NP_175971.3|transcription CK factor-related 213 0 100 gi|21280989|gb|AAM44902.1|putative PP SP catalase 214 0 89 gi|22087278|gb|AAC97995.1|Similar to LN PP HS gb|Z30094 basic transcripion factor 2,44 kD subunit from Homo sapiens. 215 0 77 gi|12324313|gb|AAD55662.1|Highly similar DS to non intermediate filament IFA bindingprotein 216 0 100 gi|21537362| gb|AAM61703.1|protein CK kinase-likeprotein 217 0 87 gi|4678332| emb|CAB41143.1|putative CK peptidetransporter 218 1.00E−144 86 gi|14532890| gb|AAK64127.1|unknown PEGprotein 219 1.00E−145 95 gi|3242721| gb|AAC23773.1|putative DSacetone-cyanohydrin lyase 220 0 84 gi|15293165| gb|AAK93693.1|putative3- CK PEG CS HS PP methyladenine DNA glycosylase 221 1.00E−178 79gi|6321581| ref|NP_011658.1|Btn2p PP [Saccharomyces cerevisiae] 222 0 98gi|30387605| ref|NP_178499.2|MATE LN efflux family protein 223 1.00E−14790 gi|9758099| ref|NP_198887.1|zinc finger CS (C2H2 type) family protein224 1.00E−139 95 gi|23505833| gb|AAN28776.1|At3g51780/ORF3 CK 225 0 100gi|6729028| gb|AAF27024.1|putative CS nodulin [Arabidopsis thaliana]ref|NP_187169.1|GDSL-motif lipase/hydrolase family protein 226 0 100gi|21436143| gb|AAM51318.1|unknown CS protein 227 0 97 gi|31711910|gb|AAM64944.1|betaine PP aldehyde dehydrogenase, putative 228 1.00E−12880 gi|21554268| ref|NP_171616.1|33 kDa PEG SS ribonucleoprotein,chloroplast, putative/RNA-binding protein cp33, putative 229 0 95gi|6321563| emb|CAA58159.1|glutamic- CK CS SS dependent asparaginesynthase 230 1.00E−132 57 gi|25403213| pir||A86468probable zinc PP PEGfinger protein 231 2.00E−54 52 gi|20127115| gb|AAM10965.1|putative CKbHLH transcription factor [Arabidopsis thaliana] 232 1.00E−160 72gi|18401370| ref|NP_566566.1|protein CK HS PEG CS phosphatase 2C familyprotein 233 1.00E−33 45 gi|21593875| gb|AAM65842.1|putative PP HSRING-H2 zinc finger protein 234 2.00E−34 51 gi|55419648|gb|AAV51937.1|AP2/EREBP CK transcription factor ERF-2 235 2.00E−73 59gi|3955021| emb|CAA09367.1|HB2 CK CS homeodomain protein 236 1.00E−51 58gi|4689366| gb|AAD27870.1|BRH1 RING HS PP PEG finger protein 2371.00E−149 76 gi|52354283| gb|AAM14913.1|putative HS malonyl-CoA:Acylcarrier protein transacylase 238 1.00E−154 85 gi|20260680|gb|AAM13238.1|putative PP PEG NADPH-dependent mannose 6-phosphatereductase 239 2.00E−89 64 gi|21593394| ref|NP_567300.1|short-chain HSdehydrogenase/reductase (SDR) family protein 240 0 82 gi|42795470|gb|AAS46245.1|HMG-CoA CK HS SS synthase 2 241 1.00E−113 83 gi|20385588|gb|AAM21344.1|MADS-box SP protein 4 242 1.00E−135 89 gi|21280889|ref|NP_196254.1|ribosomal SS HS protein S8e family protein 243 5.00E−2843 gi|4567308| gb|AAD23719.1|putative PEG RING zinc finger protein 2443.00E−35 33 gi|25352200| pir||T52379zinc finger protein PEG ZPT3-3 245 083 gi|10800918| emb|CAC12995.1|putative CK PEG CS AUX1-like permease 2463.00E−61 87 gi|21554008| gb|AAM63089.1|cold- CS HS PP regulated proteincor15b precursor 247 0 100 gi|25406415| pir||D96781 cytochrome P450, PEGprobable, 64213-66051 248 0 92 gi|3885330| gb|AAC77858.1|putative PPcytochrome P450 249 1.00E−159 100 gi|21593400| gb|AAM65367.1|phi-1-likeLN LL protein 250 1.00E−89 76 gi|45825151| ref|NP_200258.2|zinc fingerDS (B-box type) family protein 251 1.00E−144 72 gi|3643085|gb|AAC36698.1|protein PP CK phosphatase-2C; PP2C 252 2.00E−90 91gi|21537084| gb|AAM61425.1|unknown CK PEG 253 2.00E−51 100 gi|28393947|ref|NP_174092.1|glycine-rich HS PP protein 254 0 91 gi|23197704|ref|NP_565909.1|radical SAM PEG domain-containing protein 255 0 93gi|4902476| emb|CAB43520.1|MAP kinase CK CS 256 1.00E−100 72gi|34146806| gb|AAC34217.1|putative CS alcohol dehydrogenase 2572.00E−87 82 gi|3548814| gb|AAC34486.1|E3 ubiquitin CK LL CS ligase SCFcomplex subunit SKP1/ASK1 (At19), 258 1.00E−113 81 gi|7939553|dbj|BAA95756.1|expansin-like PP HS protein 259 1.00E−163 52 gi|13506810|gb|AAK28345.1|receptor-like CK PP protein kinase 3 260 1.00E−163 68gi|12597803| gb|AAG60115.1|hypothetical PP protein 261 1.00E−161 100gi|20258881| gb|AAM14112.1|putative LL ubiquinone/menaquinonebiosynthesis methyltransferase 262 1.00E−133 92 gi|12597757|ref|NP_176849.1|nodulin SS PEG MtN3 family protein 263 0 95 gi|6911875|sp|P53780|METC_ARATH PP SS Cystathionine beta-lyase, chloroplastprecursor (CBL) (Beta-cystathionase) (Cysteine lyase) 264 1.00E−100 100gi|21389647| gb|AAM48022.1|photoassimilate- SP DS responsive proteinPAR-1b- like protein 265 0 100 gi|25402889| ref|NP_173376.1|very-long-PEG PP chain fatty acid condensing enzyme, putative 266 0 88 gi|6324999|sp|P20438|CG12_YEAST DS G1/S-specific cyclin CLN2 gb|AAA65725.1|cyclin 2267 0 100 gi|3218550| dbj|BAA28775.1|Cdk- LL activating kinase 1At 2681.00E−169 100 gi|6321880| ref|NP_011956.1|Nucleolar SP protein involvedin the assembly of the large ribosomal subunit; contains asigma(70)-like motif, which is thought to bind RNA 269 0 89 gi|21436315|gb|AAM51327.1|putative CK HS CS histidyl-tRNA synthetase 270 1.00E−150100 gi|14318575| ref|NP_116708.1|20S PP PEG SS proteasome beta-typesubunit 271 0 99 gi|6325396| pir||S69027 ammonium LL DS transportprotein MEP3 272 5.00E−76 72 gi|7442240| sp|O24543|AX2E_PHAAU DSAuxin-induced protein 22E (Indole-3-acetic acid induced protein ARG14)273 0 94 gi|9759247| dbj|BAB09771.1|serine/threonine LL proteinkinase-like protein 274 0 97 gi|39647867| emb|CAE26387.1|phosphogly CKDS CS cerate kinase 275 0 89 gi|9758468| dbj|BAB08997.1|monosaccharidePP PEG transporter 276 1.00E−66 80 gi|6319966|ref|NP_010046.1|Phosphorelay HS intermediate protein, phosphorylated bythe plasma membrane sensor Sln1p in response to osmotic stress and thenin turn phosphorylates the response regulators Ssk1p in the cytosol andSkn7p in the nucleus 277 2.00E−88 83 gi|21536637| ref|NP_565524.1|stressDS LN enhanced protein 2 (SEP2) 278 0 90 gi|6322724|ref|NP_012797.1|Required for SS transcription of rDNA by RNA PolymeraseI; DNA- independent RNA Polymerase I transcription factor 279 0 94gi|6320705| pir||S69555 myo-inositol HS SS PP transport protein ITR1 -yeast) 280 5.00E−24 49 gi|25453551| pir||T52011ethylene LL responsiveelement binding factor 3 281 1.00E−150 100 gi|4580468|gb|AAD24392.1|putative PP cAMP-dependent protein kinase 282 1.00E−169 91gi|21436057| ref|NP_193037.1| LL SS oxidoreductase, zinc-bindingdehydrogenase family protein 283 1.00E−165 83 gi|21280925|gb|AAM44967.1|putative HS cinnamyl alcohol dehydrogenase 284 0 100gi|20334800| ref|NP_568453.1|alcohol PEG dehydrogenase, putative[Arabidopsis thaliana] 285 0 100 gi|22136298| gb|AAM91227.1|alcohol PPdehydrogenase 286 1.00E−144 89 gi|20259173| sp|O04202|IF35_ARATH CS SSCK Eukaryotic translation initiation factor 3 subunit 5 (elF-3 epsilon)(elF3 p32 subunit) (elF3f) 287 6.00E−74 96 gi|21593170|ref|NP_196239.1|RNA- PP binding protein, putative 288 1.00E−112 80gi|9759521| dbj|BAB10987.1|nuclear cap- PP binding protein; CBP20 2891.00E−177 95 gi|12083276| gb|AAG48797.1|putative delta CS PEG 9desaturase 290 1.00E−111 94 gi|12083264| gb|AAG48791.1|putative GTP- LNSS PP binding protein RAB11D 291 1.00E−134 91 gi|23505935|gb|AAP86673.1|26S HS SS proteasome subunit RPN12 292 3.00E−53 86gi|26452894| dbj|BAC43525.1|putative CK HS PP SS PEG CS DNA-directed RNApolymerase 14 kDa subunit AtRPAC14 293 5.00E−94 100 gi|21554412|gb|AAM63517.1|probable CK CS PEG PP glutathione peroxidase At2g31570 2941.00E−116 92 gi|6143884| ref|NP_187617.1| SP CK immunophilin, putative/FKBP-type peptidyl-prolyl cis- trans isomerase, putative 295 1.00E−114100 gi|6671929| gb|AAF23189.1|putative GTP- PP binding protein (ATFP8)[Arabidopsis thaliana] 296 1.00E−161 92 gi|7670024|ref|NP_566563.1|ubiquitin- HS PP SS conjugating enzyme, putative 2971.00E−132 95 gi|21554045| gb|AAM63126.1|20S SS proteasome subunit PAC1298 1.00E−111 94 gi|21593047| gb|AAM64996.1|GTP-binding PP protein Rab11299 0 93 gi|23308437| ref|NP_190336.1|malate SS HS dehydrogenase [NAD],chloroplast (MDH) 300 3.00E−94 80 gi|6562282| emb|CAB62652.1|rac-likePEG GTP binding protein Arad11 301 0 100 gi|21554607|gb|AAM63631.1|ubiquitin SS activating enzyme-like protein 302 5.00E−2681 gi|15217910| ref|NP_173453.1|homeobox- PP leucine zipperprotein-related 303 1.00E−129 86 gi|23505995| ref|NP_177122.2|acid SP PPphosphatase, putative [Arabidopsis thaliana] 304 1.00E−157 100gi|28827628| gb|AAO50658.1|putative C-4 PP sterol methyloxidase 3059.00E−19 100 gi|21592539| ref|NP_565794.1| CS SS CK hydroxyproline-richglycoprotein family protein [Arabidopsis thaliana] 306 0 97 gi|15965196|ref|NP 385549.1|PROBABLE SP LL ENOLASE PROTEIN 307 1.00E−153 100gi|16080137| pir||A69990 UTP-glucose-1- LL phosphate uridylyltransferasehomolog ytdA 308 1.00E−147 98 gi|26246388| ref|NP_752427.1|Pyrroline-5-LN carboxylate reductase 309 1.00E−135 94 gi|23126946|ref|ZP_00108826.1|COG0345: PP Pyrroline-5-carboxylate reductase 310 0100 gi|16263079| ref|NP_435872.1|probable PEG alcohol 311 0 99gi|15888903| pir||H97551 probable LL aminotransferase aatc 312 0 90gi|16080158| ref|NP_390984.1|glycine CS CK betaine aldehydedehydrogenase 313 1.00E−141 94 gi|15614866| ref|NP_243169.1|UTP- PPglucose-1-phosphate uridylyltransferas 314 0 99 gi|23111329|ref|ZP_00097007.1|COG0205: CS PP CK 6-phosphofructokinase 315 1.00E−15087 gi|37526393| ref|NP_929737.1|UTP-- PEG SS glucose-1-phosphateuridylyltransferase (UDP- glucose pyrophosphorylase) (UDPGP) 316 0 99gi|16128895| ref|NP_415448.1|aspartate PP aminotransferase 317 0 97gi|16080149| ref|NP_390975.1|glucose-1- DS phosphate adenylyltransferase318 0 96 gi|48732455| ref|ZP_00266198.1|COG1012: HS PP NAD-dependentaldehyde dehydrogenases 319 0 97 gi|16129263| ref|NP_415818.1|4- SS PPaminobutyrate aminotransferase 320 0 99 gi|16128583|ref|NP_415133.1|putative PP PLP-dependent aminotransferase 321 0 99gi|30063716| ref|NP_837887.1|putative CS CK aminotransferase 322 0 99gi|16130084| ref|NP_416651.1|bifunctional: SS PP putative glutamatesynthase (N-terminal); putative oxidoreductase (C-terminal) 323 0 94gi|16128664| ref|NP_415214.1|phosphoglucomutase SP PP 324 0 96gi|49176307| ref|NP_417544.3|probable CS CK ornithine aminotransferase[Escherichia coli K12] (EC 2.6.1.13) 325 1.00E−175 80 gi|48729503|ref|ZP_00263253.1|COG0508: CK PP Pyruvate/2-oxoglutarate dehydrogenasecomplex, dihydrolipoamide acyltransferase (E2) component, and relatedenzymes 326 1.00E−173 80 gi|28869402| ref|NP_792021.1|2- CK PPoxoglutarate dehydrogenase, E2 component, dihydrolipoamidesuccinyltransferase 327 1.00E−161 90 gi|37524574|ref|NP_927918.1|Transaldolase B PP 328 0 82 gi|37525385|ref|NP_928729.1|Dihydrolipoamide LN PP succinyltransferase component of2-oxoglutarate dehydrogenase complex (E2) 329 3.00E−55 86 gi|16332334|ref|NP_443062.1|hypothetical CS PP HS protein slr0607 330 0 100gi|15614388| ref|NP_242691.1|acetoin LL dehydrogenase E3 component 331 099 gi|15615327| ref|NP_243630.1|dihydrolipoamide LN dehydrogenase 332 097 gi|16078525| ref|NP_389344.1|dihydrolipoamide HS DS dehydrogenase E3subunit of both pyruvate dehydrogenase and 2- oxoglutarate dehydrogenasecomplexes 333 0 94 gi|15800431| ref|NP_286443.1|2- CS CK oxoglutaratedehydrogenase 334 0 89 gi|22136798| gb|AAM91743.1|putative LNphosphate/phosphoenolpyruvate translocator precursor protein 335 0 100gi|49176098| ref|NP_415825.3|putative HS SS polysaccharide hydrolase 3360 96 gi|15889444| ref|NP_355125.1|AGR_C_3927p CK HS PP PEG CS SS[Agrobacterium tumefaciens str. C58] ref|NP_532838.1|bacteriophytochrome protein 337 0 95 gi|15613173|ref|NP_241476.1|sulfite PP CK CS PEG reductase (NADPH) 338 0 100gi|30062749| ref|NP_836920.1|nitrate LN reductase 1, beta subunit 339 0100 gi|15804618| ref|NP_290659.1|glucosephosphate PP CS isomerase 340 0100 gi|23125493| ref|ZP_00107424.1|COG0243: PP PEG HS Anaerobicdehydrogenases, typically selenocysteine- containing 341 0 98gi|28868179| ref|NP_790798.1|glucose-6- CK PP SP PEG CS phosphateisomerase 342 0 96 gi|16329427| ref|NP_440155.1|isocitrate CK CSdehydrogenase (NADP+) 343 0 85 gi|37524705| ref|NP_928049.1|sulfite DSPP PEG reductase [NADPH] hemoprotein beta-component (SIR-HP) 3441.00E−121 95 gi|6728966| gb|AAF26964.1|unknown LN protein 345 0 96gi|6714417| gb|AAF26105.1|unknown LN protein 346 0 78 gi|22136866|ref|NP_177343.2|protease- CS associated zinc finger (C3HC4-type RINGfinger) family protein 347 0 92 gi|51971567| ref|NP_850943.1|glutamineCK HS CS amidotransferase-related 348 2.00E−65 88 gi|26450572|dbj|BAC42398.1|unknown CK CS protein [Arabidopsis thaliana] 3491.00E−115 100 gi|6730712| gb|AAF27107.1|Unknown CK CS protein 350 0 100gi|20465757| gb|AAM20367.1|putative PP cyclin protein 351 8.00E−90 56gi|23297314| ref|NP_849559.1|WRKY CK CS family transcription factor[Arabidopsis thaliana] 352 1.00E−171 83 gi|50940357|ref|XP_479706.1|putative PP HS SS Shwachman-Bodian-Diamond syndromeprotein 353 2.00E−46 92 gi|50929801| ref|XP_474428.1|OSJNBa0070M12.6 CKPP 354 7.00E−54 92 gi|5042333| emb|CAB44664.1|BETL4 PP protein 3551.00E−56 34 gi|42563228| ref|NP_565108.2|zinc finger HS SS PEG(CCCH-type) family protein 356 4.00E−61 72 gi|51970440|dbj|BAD43912.1|hypothetical CK PP protein 357 7.00E−91 99 gi|16331395|ref|NP_442123.1|hypothetical SS protein slr0013 358 0 99 gi|21229841|ref|NP_635758.1|vanillate O- CS CK demethylase oxygenase subunit 3591.00E−142 100 gi|16331855| sp|Q55891|PCYA_SYNY3 CK PP CSPhycocyanobilin:ferredoxin oxidoreductase 360 0 100 gi|16331872|ref|NP_442600.1|hypothetical SP CK protein slr0304 361 3.00E−86 87gi|21593344| gb|AAM65293.1|putative cold- LN regulated proteinref|NP_178469.1|late embryogenesis abundant domain-containing protein/LEA domain-containing protein 362 8.00E−91 100 gi|16330328|sp|P73690|Y51L_SYNY3 HS Ycf51-like protein dbj|BAA17736.1| ORF_ID:sII1702~hypothetical protein 363 5.00E−87 100 gi|15612647|ref|NP_240950.1|hypoxanthine- PEG SS guanine phosphoribosyltransferase364 0 83 gi|6323679| sp|P23748|MPIP_YEAST M- CS LL PP CK HS SS phaseinducer phosphatase (Mitosis initiation protein MIH1) (Mitotic inducerhomolog) 365 2.00E−33 90 gi|34898476| ref|NP_910584.1|ESTAU082567 PP(S21715) corresponds to a region of the predicted gene.~Similar to S.tuberosum ubiquinol cytochrome c reductase. (X79275) 366 6.00E−54 81gi|21592528| gb|AAM64477.1|ring-box HS SS protein-like 367 0 76gi|34910110| dbj|BAB92553.1|DNA cross-link CK HS repair 1B-like protein368 1.00E−156 100 gi|21436267| gb|AAM51272.1|putative LN nodulin-26protein 369 1.00E−100 81 gi|50900588| ref|XP_462727.1|putative LNphenylalkylamine binding protein sp|Q9FTZ2|EBP_ORYSA Probable 3-beta-hydroxysteroid- delta(8),delta(7)-isomerase (Cholestenoldelta-isomerase) (Delta8-delta7 sterol isomerase) (D8-D7 sterolisomerase) dbj|BAB92148.1| putative C-8,7 sterol isomerase 370 4.00E−3946 gi|25361093| pir||T00967hypothetical HS protein At2g26340 3712.00E−78 89 gi|50906887| ref|XP_464932.1|cytochrome LL PP c biogenesisprotein-like 372 1.00E−22 98 gi|50899510| ref|XP_450543.1|unknown CK LLCS PP SS protein 373 5.00E−79 85 gi|50934647|ref|XP_476851.1|bifunctional CK CS phosphopantetheine adenylyltransferase dephospho CoA kinase-like protein 374 8.00E−56 50gi|38257027| dbj|BAD01556.1|ERF-like PP protein 375 1.00E−64 55gi|18423944| ref|NP_568850.1|basic helix- PP loop-helix (bHLH) familyprotein 376 1.00E−39 42 gi|42567912| ref|NP_568344.2|myb family SP PEGtranscription factor 377 6.00E−89 84 gi|37535020|ref|NP_921812.1|putative LN HAM-1-like protein 378 2.00E−49 59gi|27804371| gb|AAO22987.1|MADS-box LL SS transcription factor CDM104379 2.00E−68 60 gi|22137112| emb|CAB72174.1|responce CK SS reactor 4[Arabidopsis thaliana] 380 0 89 gi|50910245| ref|XP_466611.1|putative CSCK PLRR-4 polymorphic leucine- rich repeat protein 381 2.00E−30 44gi|17933450| gb|AAK70215.1|MADS-box CS CK PEG protein 382 1.00E−93 54gi|20502508| dbj|BAB91414.1|E2F-like CK PEG CS repressor E2L3 3832.00E−38 58 gi|50909627| ref|XP_466302.1|unknown PEG protein 3843.00E−41 63 gi|50399946| gb|AAT76334.1|putative DNA- HS directed RNApolymerase II subunit 385 4.00E−59 68 gi|37535924|ref|NP_922264.1|unknown LN protein 386 8.00E−57 58 gi|50944571|ref|XP_481813.1|transfactor- HS like 387 3.00E−73 42 gi|53792319|dbj|BAD53026.1|putative ring LN finger protein 1 388 9.00E−93 47gi|25054862| ref|NP_850517.1| LN transcription factor, putative/ zincfinger (C3HC4 type RING finger) family protein 389 7.00E−91 66gi|20269059| emb|CAC84710.1|aux/IAA PEG protein 390 2.00E−72 45gi|26450026| ref|NP_172358.1|myb family LL LN transcription factor(MYB60) 391 1.00E−80 46 gi|50946213| ref|XP_482634.1|AP2/EREBP LNtranscription factor-like protein 392 8.00E−54 43 gi|29824137|ref|NP_189337.1|TCP family CK CS transcription factor, putative[Arabidopsis thaliana] 393 0 99 gi|558543| emb|CAA85320.1|C-terminal HSPP PEG zinc-finger 394 1.00E−124 62 gi|20259301| ref|NP_566010.1|SET PPSS domain-containing protein (ASHH3) 395 4.00E−54 65 gi|21553740|gb|AAM62833.1|putative zinc PP PEG CS finger protein 396 2.00E−54 40gi|20465561| ref|NP_974448.1|zinc finger LL LN (C3HC4-type RING finger)family protein 397 1.00E−112 65 gi|51557078| gb|AAU06309.1|MYB PP LNtranscription factor 398 0 82 gi|15148926| gb|AAK84890.1|TGA-type LNbasic leucine zipper protein TGA2.2 399 6.00E−78 47 gi|28558782|gb|AAO45753.1|RING/C3HC4/ LN PHD zinc finger-like protein 400 0 67gi|42565068| ref|NP_188743.3|transducin HS family protein/WD-40 repeatfamily protein 401 7.00E−29 46 gi|38638682| ref|NP_177307.1|AP2 CS LL LNdomain-containing transcription factor, putative 402 0 84 gi|50904461|ref|XP_463719.1|P0466H10.27 PEG 403 0 88 gi|53796982|ref|ZP_00357872.1|COG0160: SP CS 4-aminobutyrate aminotransferase andrelated aminotransferases 404 0 94 gi|53796007|ref|ZP_00357032.1|COG0696: PP SS PEG HS Phosphoglyceromutase 405 0 93gi|30697938| ref|NP_201207.2|expressed DS HS protein 406 0 100gi|13878095| gb|AAK44125.1|unknown DS CS protein 407 0 97 gi|12324320|gb|AAG52129.1|hypothetical LL protein; 63994-65574 408 0 84 gi|22136086|gb|AAM91121.1|photoreceptor- CS PP interacting protein-like

Trait Improvement Screens

DS-Improvement of Drought Tolerance Identified by a Soil Drought StressTolerance Screen:

Drought or water deficit conditions impose mainly osmotic stress onplants. Plants are particularly vulnerable to drought during theflowering stage. The drought condition in the screening processdisclosed in Example 1B started from the flowering time and wassustained to the end of harvesting. The present invention providesrecombinant DNA that can improve the plant survival rate under suchsustained drought condition. Exemplary recombinant DNA for conferringsuch drought tolerance are identified as such in Table 3. Suchrecombinant DNA may find particular use in generating transgenic plantsthat are tolerant to the drought condition imposed during flowering timeand in other stages of the plant life cycle. As demonstrated from themodel plant screen, in some embodiments of transgenic plants withtrait-improving recombinant DNA grown under such sustained droughtcondition can also have increased total seed weight per plant inaddition to the increased survival rate within a transgenic population,providing a higher yield potential as compared to control plants.

PEG-Improvement of Drought Tolerance Identified by PEG Induced OsmoticStress Tolerance Screen:

Various drought levels can be artificially induced by using variousconcentrations of polyethylene glycol (PEG) to produce different osmoticpotentials (Pilon-Smits et al. (1995) Plant Physiol. 107:125-130).Several physiological characteristics have been reported as beingreliable indications for selection of plants possessing droughttolerance. These characteristics include the rate of seed germinationand seedling growth. The traits can be assayed relatively easily bymeasuring the growth rate of seedling in PEG solution. Thus, aPEG-induced osmotic stress tolerance screen is a useful surrogate fordrought tolerance screen. As demonstrated from the model plant screen,embodiments of transgenic plants with trait-improving recombinant DNAidentified in the PEG-induced osmotic stress tolerance screen cansurvive better drought conditions providing a higher yield potential ascompared to control plants.

SS-Improvement of Drought Tolerance Identified by High Salinity StressTolerance Screen:

Three different factors are responsible for salt damages: (1) osmoticeffects, (2) disturbances in the mineralization process, (3) toxiceffects caused by the salt ions, e.g. inactivation of enzymes. While thefirst factor of salt stress results in the wilting of the plants that issimilar to drought effect, the ionic aspect of salt stress is clearlydistinct from drought. The present invention provides genes that helpplants to maintain biomass, root growth, and/or plant development inhigh salinity conditions, which are identified as such in Table 3. Sinceosmotic effect is one of the major components of salt stress, which iscommon to the drought stress, trait-improving recombinant DNA identifiedin a high salinity stress tolerance screen can also provide transgeniccrops with improved drought tolerance. As demonstrated from the modelplant screen, embodiments of transgenic plants with trait-improvingrecombinant DNA identified in a high salinity stress tolerance screencan survive better drought conditions and/or high salinity conditionsproviding a higher yield potential as compared to control plants.

HS-Improvement of Drought Tolerance Identified by Heat Stress ToleranceScreen:

Heat and drought stress often occur simultaneously, limiting plantgrowth. Heat stress can cause the reduction in photosynthesis rate,inhibition of leaf growth and osmotic potential in plants. Thus, genesidentified by the present invention as heat stress tolerance conferringgenes may also impart improved drought tolerance to plants. Asdemonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in a heat stresstolerance screen can survive better heat stress conditions and/ordrought conditions providing a higher yield potential as compared tocontrol plants.

CK and CS-Improvement of Tolerance to Cold Stress:

Low temperature may immediately result in mechanical constraints,changes in activities of macromolecules, and reduced osmotic potential.In the present invention, two screening conditions, i.e. cold shocktolerance screen (CK) and cold germination tolerance screen (CS), wereset up to look for transgenic plants that display visual growthadvantage at lower temperature. In cold germination tolerance screen,the transgenic Arabidopsis plants were exposed to a constant temperatureof 8° C. from planting until day 28 post plating. The trait-improvingrecombinant DNA identified by such screen are particular useful for theproduction of transgenic plant that can germinate more robustly in acold temperature as compared to the wild type plants. In cold shocktolerance screen, the transgenic plants were first grown under thenormal growth temperature of 22° C. until day 8 post plating, andsubsequently were placed under 8° C. until day 28 post plating. Asdemonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in a cold shockstress tolerance screen and/or a cold germination stress tolerancescreen can survive better cold conditions providing a higher yieldpotential as compared to control plants.

Improvement of Tolerance to Multiple Stresses:

Different kinds of stresses often lead to identical or similar reactionin the plants. Genes that are activated or inactivated as a reaction tostress can either act directly in a way the genetic product reduces aspecific stress, or they can act indirectly by activating other specificstress genes. By manipulating the activity of such regulatory genes,i.e. multiple stress tolerance genes, the plant can be enabled to reactto different kinds of stresses. For examples, PEP SEQ ID NO: 229 and PEPSEQ ID NO: 372 can be used to improve both salt stress tolerance andcold stress tolerance in plants. Of particular interest, plantstransformed with PEP SEQ ID NO: 364 can resist heat stress, salt stressand cold stress. In addition to these multiple stress tolerance genes,the stress tolerance conferring genes provided by the present inventionmay be used in combinations to generate transgenic plants that canresist multiple stress conditions.

PP-Improvement of Early Plant Growth and Development:

It has been known in the art that to minimize the impact of disease oncrop profitability, it is important to start the season with healthyvigorous plants. This means avoiding seed and seedling diseases, leadingto increased nutrient uptake and increased yield potential.Traditionally early planting and applying fertilizer are the methodsused for promoting early seedling vigor. In early development stage,plant embryos establish only the basic root-shoot axis, a cotyledonstorage organ(s), and stem cell populations, called the root and shootapical meristems, that continuously generate new organs throughoutpost-embryonic development. “Early growth and development” used hereinencompasses the stages of seed imbibition through the early vegetativephase. The present invention provides genes that are useful to producetransgenic plants that have advantages in one or more processesincluding, but not limited to, germination, seedling vigor, root growthand root morphology under non-stressed conditions. The transgenic plantsstarting from a more robust seedling are less susceptible to the fungaland bacterial pathogens that attach germinating seeds and seedling.Furthermore, seedlings with advantage in root growth are more resistantto drought stress due to extensive and deeper root architecture.Therefore, it can be recognized by those skilled in the art that genesconferring the growth advantage in early stages to plants may also beused to generate transgenic plants that are more resistant to variousstress conditions due to improved early plant development. The presentinvention provides such exemplary recombinant DNA that confer both thestress tolerance and growth advantages to plants, identified as such inTable 3, e.g. PEP SEQ ID NO: 372 which can improve the plant earlygrowth and development, and impart salt and cold tolerance to plants. Asdemonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in the earlyplant development screen can grow better under non-stress conditionsand/or stress conditions providing a higher yield potential as comparedto control plants.

SP-Improvement of Late Plant Growth and Development:

“Late growth and development” used herein encompasses the stages of leafdevelopment, flower production, and seed maturity. In certainembodiments, transgenic plants produced using genes that confer growthadvantages to plants provided by the present invention, identified assuch in Table 3, exhibit at least one phenotypic characteristicsincluding, but not limited to, increased rosette radius, increasedrosette dry weight, seed dry weight, silique dry weight, and siliquelength. On one hand, the rosette radius and rosette dry weight are usedas the indexes of photosynthesis capacity, and thereby plant sourcestrength and yield potential of a plant. On the other hand, the seed dryweight, silique dry weight and silique length are used as the indexesfor plant sink strength, which are considered as the direct determinantsof yield. As demonstrated from the model plant screen, embodiments oftransgenic plants with trait-improving recombinant DNA identified in thelate development screen can grow better and/or have improved developmentduring leaf development and seed maturation providing a higher yieldpotential as compared to control plants.

LL-Improvement of Tolerance to Shade Stress Identified in a Low LightScreen:

The effects of light on plant development are especially prominent atthe seedling stage. Under normal light conditions with unobstructeddirect light, a plant seeding develops according to a characteristicphotomorphogenic pattern, in which plants have open and expandedcotyledons and short hypocotyls. Then the plant's energy is devoted tocotyledon and leaf development while longitudinal extension growth isminimized. Under low light condition where light quality and intensityare reduced by shading, obstruction or high population density, aseedling displays a shade-avoidance pattern, in which the seedlingdisplays a reduced cotyledon expansion, and hypocotyls extension isgreatly increased. As the result, a plant under low light conditionincreases significantly its stem length at the expanse of leaf, seed orfruit and storage organ development, thereby adversely affecting ofyield. The present invention provides recombinant DNA that enable plantsto have an attenuated shade avoidance response so that the source ofplant can be contributed to reproductive growth efficiently, resultinghigher yield as compared to the wild type plants. As demonstrated fromthe model plant screen, embodiments of transgenic plants withtrait-improving recombinant DNA identified in a shade stress tolerancescreen can have attenuated shade response under shade conditionsproviding a higher yield potential as compared to control plants. Thetransgenic plants generated by the present invention may be suitable fora higher density planting, thereby resulting increased yield per unitarea.

LN-Improvement of Tolerance to Low Nitrogen Availability Stress

Nitrogen is a key factor in plant growth and crop yield. The metabolism,growth and development of plants are profoundly affected by theirnitrogen supply. Restricted nitrogen supply alters shoot to root ratio,root development, activity of enzymes of primary metabolism and the rateof senescence (death) of older leaves. All field crops have afundamental dependence on inorganic nitrogenous fertilizer. Sincefertilizer is rapidly depleted from most soil types, it must be suppliedto growing crops two or three times during the growing season. Enhancednitrogen use efficiency by plants should enable crops cultivated underlow nitrogen availability stress condition resulted from low fertilizerinput or poor soil quality.

According to the present invention, transgenic plants generated usingthe recombinant nucleotides, which confer enhanced nitrogen useefficiency, identified as such in Table 3, exhibit one or more desirabletraits including, but not limited to, increased seedling weight,increased number of green leaves, increased number of rosette leaves,increased root length and advanced flower bud formation. One skilled inthe art may recognize that the transgenic plants provided by the presentinvention with enhanced nitrogen use efficiency may also have alteredamino acid or protein compositions, increased yield and/or better seedquality. The transgenic plants of the present invention may beproductively cultivated under nitrogen nutrient deficient conditions,i.e. nitrogen-poor soils and low nitrogen fertilizer inputs, that wouldcause the growth of wild type plants to cease or to be so diminished asto make the wild type plants practically useless. The transgenic plantsalso may be advantageously used to achieve earlier maturing, fastergrowing, and/or higher yielding crops and/or produce more nutritiousfoods and animal feedstocks when cultivated using nitrogen non-limitinggrowth conditions.

Stacked Traits:

The present invention also encompasses transgenic plants with stackedengineered traits, e.g. a crop having an improved phenotype resultingfrom expression of a trait-improving recombinant DNA, in combinationwith herbicide and/or pest resistance traits. For example, genes of thecurrent invention can be stacked with other traits of agronomicinterest, such as a trait providing herbicide resistance, for example aRoundUp Ready trait, or insect resistance, such as using a gene fromBacillus thuringensis to provide resistance against lepidopteran,coliopteran, homopteran, hemiopteran, and other insects. Herbicides forwhich resistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides andgluphosinate herbicides. To illustrate that the production of transgenicplants with herbicide resistance is a capability of those of ordinaryskill in the art, reference is made to U.S. patent applicationpublications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos.5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which areincorporated herein by reference. To illustrate that the production oftransgenic plants with pest resistance is a capability of those ofordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515and 5,880,275 which disclose plants expressing an endotoxin of Bacillusthuringiensis bacteria, to U.S. Pat. No. 6,506,599 which disclosescontrol of invertebrates which feed on transgenic plants which expressdsRNA for suppressing a target gene in the invertebrate, to U.S. Pat.No. 5,986,175 which discloses the control of viral pests by transgenicplants which express viral replicase, and to U.S. Patent ApplicationPublication 2003/0150017 A1 which discloses control of pests by atransgenic plant which express a dsRNA targeted to suppressing a gene inthe pest, all of which are incorporated herein by reference.

Once one recombinant DNA has been identified as conferring an improvedtrait of interest in transgenic Arabidopsis plants, several methods areavailable for using the sequence of that recombinant DNA and knowledgeabout the protein it encodes to identify homologs of that sequence fromthe same plant or different plant species or other organisms, e.g.bacteria and yeast. Thus, in one aspect, the invention provides methodsfor identifying a homologous gene with a DNA sequence homologous to anyof SEQ ID NO: 1 through SEQ ID NO: 204, or a homologous protein with anamino acid sequence homologous to any of SEQ ID NO: 205 through SEQ IDNO: 408. In another aspect, the present invention provides the proteinsequences of identified homologs for a sequence listed as SEQ ID NO: 205through SEQ ID NO: 408. In yet another aspect, the present inventionalso includes linking or associating one or more desired traits, or genefunction with a homolog sequence provided herein.

The trait-improving recombinant DNA and methods of using suchtrait-improving recombinant DNA for generating transgenic plants withimproved traits provided by the present invention are not limited to anyparticular plant species. Indeed, the plants according to the presentinvention may be of any plant species, i.e., may be monocotyledonous ordicotyledonous. Preferably, they will be agricultural useful plants,i.e., plants cultivated by man for purposes of food production ortechnical, particularly industrial applications. Of particular interestin the present invention are corn and soybean plants. The recombinantDNA constructs optimized for soybean transformation and recombinant DNAconstructs optimized for corn transformation are provided by the presentinvention. Other plants of interest in the present invention forproduction of transgenic plants having improved traits include, withoutlimitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley,millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

In certain embodiments, the present invention contemplates to use anorthologous gene in generating the transgenic plants with similarlyimproved traits as the transgenic Arabidopsis counterpart. Improvedphysiological properties in transgenic plants of the present inventionmay be confirmed in responses to stress conditions, for example inassays using imposed stress conditions to detect improved responses todrought stress, nitrogen deficiency, cold growing conditions, oralternatively, under naturally present stress conditions, for exampleunder field conditions. Biomass measures may be made on greenhouse orfield grown plants and may include such measurements as plant height,stem diameter, root and shoot dry weights, and, for corn plants, earlength and diameter.

Trait data on morphological changes may be collected by visualobservation during the process of plant regeneration as well as inregenerated plants transferred to soil. Such trait data includescharacteristics such as normal plants, bushy plants, taller plants,thicker stalks, narrow leaves, striped leaves, knotted phenotype,chlorosis, albino, anthocyanin production, or altered tassels, ears orroots. Other enhanced traits may be identified by measurements takenunder field conditions, such as days to pollen shed, days to silking,leaf extension rate, chlorophyll content, leaf temperature, stand,seedling vigor, internode length, plant height, leaf number, leaf area,tillering, brace roots, stay green, stalk lodging, root lodging, planthealth, barreness/prolificacy, green snap, and pest resistance. Inaddition, trait characteristics of harvested grain may be confirmed,including number of kernels per row on the ear, number of rows ofkernels on the ear, kernel abortion, kernel weight, kernel size, kerneldensity and physical grain quality.

To confirm hybrid yield in transgenic corn plants expressing genes ofthe present invention, it may be desirable to test hybrids over multipleyears at multiple locations in a geographical location where maize isconventionally grown, e.g. in Iowa, Illinois or other locations in themidwestern United States, under “normal” field conditions as well asunder stress conditions, e.g. under drought or population densitystress.

Transgenic plants can be used to provide plant parts according to theinvention for regeneration or tissue culture of cells or tissuescontaining the constructs described herein. Plant parts for thesepurposes can include leaves, stems, roots, flowers, tissues, epicotyl,meristems, hypocotyls, cotyledons, pollen, ovaries, cells andprotoplasts, or any other portion of the plant which can be used toregenerate additional transgenic plants, cells, protoplasts or tissueculture. Seeds of transgenic plants are provided by this invention canbe used to propagate more plants containing the trait-improvingrecombinant DNA constructs of this invention. These descendants areintended to be included in the scope of this invention if they contain atrait-improving recombinant DNA construct of this invention, whether ornot these plants are selfed or crossed with different varieties ofplants.

The various aspects of the invention are illustrated by means of thefollowing examples which are in no way intended to limit the full breathand scope of claims.

Example 1

This example illustrates the identification of recombinant DNA thatconfers improved trait(s) to plants

A large set of genes of interest were cloned from a genomic or cDNAlibrary using primers specific to sequences upstream and downstream ofthe coding region. Transformation vectors were prepared toconstitutively transcribe DNA in either sense orientation (for enhancedprotein expression) or anti-sense orientation (for endogenous genesuppression) under the control of an enhanced Cauliflower Mosaic Virus35S promoter. The transformation vectors also contain a bar gene as aselectable marker for resistance to glufosinate herbicide. Thetransformation of Arabidopsis plants was carried out using the vacuuminfiltration method known in the art (Bethtold et al. Methods Mol. Biol.82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds,were subsequently grown in a glufosinate-containing selective medium toselect for plants which were actually transformed and which produced T2transgenic seed. The plants and seeds were screened for an enhancedtrait or a surrogate for an enhanced trait.

Soil Drought Tolerance Screen

This screen identified genes for recombinant DNA that imparts enhancedwater use efficiency as shown in Arabidopsis plants transformed withrecombinant DNA that wilt less rapidly and/or produce higher seed yieldwhen grown in soil under drought conditions

T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts®Company, USA). Humidity domes were added to each flat and flats wereassigned locations and placed in climate-controlled growth chambers.Plants were grown under a temperature regime of 22° C. at day and 20° C.at night, with a photoperiod of 16 hours and average light intensity of170 μmol/m²/s. After the first true leaves appeared, humidity domes wereremoved. The plants were sprayed with glufosinate herbicide and put backin the growth chamber for 3 additional days. Flats were watered for 1hour the week following the herbicide treatment. Watering was continuedevery seven days until the flower bud primordia became apparent, atwhich time plants were watered for the last time.

To identify drought tolerant plants, plants were evaluated for wiltingresponse and seed yield. Beginning ten days after the last watering,plants were examined daily until 4 plants/line had wilted. In the nextsix days, plants were monitored for wilting response. Five droughtscores were assigned according to the visual inspection of thephenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severewilting, and 5 for dead. A score of 3 or higher was considered aswilted.

At the end of this assay, seed yield measured as seed weight per plantunder the drought condition was characterized for the transgenic plantsand their controls and analyzed as a quantitative response according toexample 1M.

Two approaches were used for statistical analysis on the wiltingresponse. First, the risk score was analyzed for wilting phenotype andtreated as a qualitative response according to the example 1L.Alternatively, the survival analysis was carried out in which theproportions of wilted and non-wilted transgenic and control plants werecompared over each of the six days under scoring and an overall log ranktest was performed to compare the two survival curves using S-PLUSstatistical software (S-PLUS 6, Guide to statistics, Insightful,Seattle, Wash., USA). Table 4 provides a list of recombinant DNAconstructs that improve drought tolerance in transgenic plants.

TABLE 4 Wilt Response Seed Survival Anaysis of Pep Risk scoreWeight/plant wilt response SEQ Con- Orien- RS p- p- diff time p- IDstruct_id tation mean value c delta value c to wilting value c 215 19116ANTI- 0 0.795 S −0.341 0.795 / 0.52 0.077 T SENSE 250 70677 SENSE 0.0450.982 S −5.73 0.982 / 0.14 0.07 T 219 72712 SENSE 0.002 0.986 S −2.8220.986 / 0.64 0.242 / 266 72714 SENSE 0.01 0 S 1.253 0 S 0.14 0.059 T 27172721 SENSE 0.05 0.834 T −0.129 0.834 / 0 1 / 272 72959 SENSE 0.0010.172 S 0.341 0.172 / 0.42 0.32 / 317 73534 SENSE 0.006 0.732 S −0.1950.732 / −0.14 0.724 / 264 74244 SENSE 0.012 0.984 S −0.512 0.984 / 0.130.54 / 332 74453 SENSE 0.655 0.001 / 0.886 0.001 S −0.15 0.468 / S:represents that the transgenic plants showed statistically significanttrait improvement as compared to the reference (p < 0.05, p value, ofthe delta of a quantitative response or of the risk score of aqualitative response, is the probability that the observed differencebetween the transgenic plants and the reference occur by chance) T:represents that the transgenic plants showed a trend of traitimprovement as compared to the reference with p < 0.2 /: represents thetransgenic plants didn't show any alteration or had unfavorable changein traits examined compared to the reference in the current dataset.

Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and rootgrowth is inhibited. This screen identified genes for recombinant DNAthat imparts enhanced heat tolerance as shown in Arabidopsis plantstransformed with the gene of interest that are more resistant to heatstress based on primarily their seedling weight and root growth underhigh temperature.

T2 seeds were plated on ½×MS salts, 11% phytagel, with 10 μg/ml BASTA (7per plate with 2 control seeds; 9 seeds total per plate). Plates wereplaced at 4° C. for 3 days to stratify seeds. Plates were then incubatedat room temperature for 3 hours and then held vertically for 11additional days at temperature of 34° C. at day and 20° C. at night.Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After14 days of growth, plants were scored for glufosinate resistance, rootlength, final growth stage, visual color, and seedling fresh weight. Aphotograph of the whole plate was taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final grow stage at day 14 wasscored as success if 50% of the plants had reached 3 rosette leaves andsize of leaves are greater than 1 mm (Boyes et al. (2001) The Plant Cell13, 1499-1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. Table 5 provides a list of recombinantDNA constructs that improve heat tolerance in transgenic plants.

TABLE 5 Pep seedling weight Root Length growth stage SEQ Con- Orien- p-p- RS p- ID struct_id tation delta value c delta value c mean value c258 10903 ANTI- 1.26 0 S 0.131 0.053 T 0.86 0.051 T SENSE 205 12360SENSE 1.305 0 S 0.142 0.052 T 0.307 0.043 S 405 12824 SENSE 1.309 0 S0.25 0.002 S 0.63 0.063 T 211 12927 SENSE 1.527 0 S 0.268 0.013 S 0.8030.018 S 207 15210 SENSE 1.189 0 S 0.121 0.029 S 0.636 0.079 T 214 17309SENSE 1.29 0 S 0.176 0.01 S 0.581 0.044 S 242 19801 SENSE 1.156 0 S0.148 0.041 S 0.413 0.141 T 233 19845 SENSE 1.242 0 S 0.148 0.051 T0.479 0.016 S 239 19850 SENSE 1.234 0 S 0.217 0.015 S 1.883 0.001 S 23719981 SENSE 1.406 0 S 0.36 0 S 2.199 0 S 220 71546 SENSE 1.496 0 S 0.240.016 S 0.313 0.052 T 246 71556 SENSE 1.394 0 S 0.26 0.015 S 0.194 0.076T 276 73029 SENSE 0.88 0.002 S 0.115 0.061 T 0.092 0.105 T 318 74125SENSE 0.988 0 S 0.171 0.058 T 0.206 0.053 T 283 74329 SENSE 1.19 0 S0.124 0.08 T 0.043 0.239 / 329 74402 SENSE 1.407 0 S 0.291 0.019 S 0.1810.087 T 335 74503 SENSE 1.159 0 S 0.171 0.019 S 0.206 0.02 S 340 74553SENSE 1.141 0 S 0.184 0.02 S 0.704 0.03 S 299 74669 SENSE 1.105 0 S0.133 0.049 S 0.051 0.362 / 352 74903 SENSE 1.167 0 S 0.168 0.096 T0.836 0.041 S 362 74980 SENSE 1.084 0 S 0.165 0.027 S 1.07 0.033 S 36475337 SENSE 1.695 0 S 0.216 0.009 S 0.369 0.072 T 367 75352 SENSE 1.3420 S 0.198 0.003 S 0.311 0.053 T 370 75358 SENSE 1.314 0 S 0.131 0.034 S0.012 0.365 / 384 75409 SENSE 1.264 0 S 0.172 0.041 S 0.716 0.039 S 38675550 SENSE 1.117 0 S 0.18 0.032 S 0.384 0.074 T 400 75571 SENSE 1.182 0S 0.185 0.042 S 0.496 0.088 T 404 75909 SENSE 1.264 0 S 0.237 0.021 S−0.01 1 / S: represents the transgenic plants showed statisticallysignificant trait improvement as compared to the reference (p < 0.05) T:represents the transgenic plants showed a trend of trait improvement ascompared to the reference with p < 0.2 /: represents the transgenicplants didn't show any alteration or had unfavorable change in traitsexamined compared to the reference in the current dataset

Salt Stress Tolerance Screen

This screen identified genes for recombinant DNA that imparts enhancedsalt tolerance, a surrogate for enhanced water use efficiency, as shownin Arabidopsis plants transformed with the gene of interest that aretolerant to high levels of salt based on their rate of development, rootgrowth and chlorophyll accumulation under high salt conditions.

T2 seeds were plated on glufosinate selection plates containing 90 mMNaCl and grown under standard light and temperature conditions. Allseedlings used in the experiment were grown at a temperature of 22° C.at day and 20° C. at night, a 16-hour photoperiod, an average lightintensity of approximately 120 umol/m². On day 11, plants were measuredfor primary root length. After 3 more days of growth (day 14), plantswere scored for transgenic status, primary root length, growth stage,visual color, and the seedlings were pooled for fresh weightmeasurement. A photograph of the whole plate was also taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final growth stage at day 14 wasscored as success if 50% of the plants reached 3 rosette leaves and sizeof leaves are greater than 1 mm (Boyes, D. C. et. al. (2001), The PlantCell 13, 1499/1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. Table 6 provides a list of recombinantDNA constructs that improve high salinity tolerance in transgenic plants

TABLE 6 Seedling Weight Root Length Root Length Pep Con- at day 14 atday 11 at day 14 Growth Stage SEQ struct Orien- p- p- p- RS p- ID idtation delta value c delta value c delta value c mean vallue c 228 19617SENSE 0.662 0.023 S 0.211 0.138 T 0.019 0.845 / 0.467 0.06 T 229 19750SENSE 0.794 0.001 S 0.324 0.035 S 0.192 0.001 S 3.409 0.001 S 240 19942SENSE 0.496 0.022 S 0.123 0.294 / 0.103 0.208 / 1.302 0.103 T 270 72743SENSE 0.522 0.043 S 0.018 0.856 / 0.194 0.004 S 0.651 0.055 T 278 72920SENSE 0.377 0.033 S 0.251 0.001 S 0.133 0.004 S 0.191 0.233 / 315 73516SENSE 0.41 0.005 S 0.079 0.292 / 0.034 0.372 / 1.252 0.053 T 263 74237SENSE 0.711 0.002 S 0.18 0.135 T 0.187 0.066 T 2.74 0.01 S 282 74327SENSE 0.554 0.006 S 0.169 0.056 T 0.162 0.005 S 1.469 0.005 S 291 74366SENSE 0.623 0.008 S 0.125 0.228 / 0.108 0.248 / 0.695 0.062 T 335 74503SENSE 0.666 0.002 S 0.258 0.027 S 0.103 0.221 / 2.431 0.01 S 336 74504SENSE 0.976 0.001 S 0.356 0.013 S 0.261 0 S 3.178 0.001 S 296 74622SENSE 1.092 0.003 S 0.261 0.005 S 0.217 0.013 S 1.457 0.065 T 297 74628SENSE 0.423 0.057 T −0.062 0.725 / 0.226 0.02 S 0.087 0.31 / 301 74647SENSE 0.161 0.442 / −0.083 0.569 / 0.208 0.001 S 0.573 0.043 S 352 74903SENSE 0.446 0.009 S 0.001 0.99 / 0.116 0.034 S 1.234 0.05 T 357 74977SENSE 0.496 0.019 S 0.2 0.012 S 0.198 0 S 0.764 0.043 S 363 74993 SENSE0.379 0.017 S 0.242 0.075 T 0.108 0.07 T 0.163 0.319 / 366 75316 SENSE0.475 0.078 T 0.287 0.002 S 0.178 0.011 S 4 0 S 364 75337 SENSE 0.9340.004 S 0.217 0.124 T 0.314 0.002 S 1.831 0.013 S 378 75431 SENSE 0.4290.083 T 0.076 0.397 / 0.096 0.271 / 0.522 0.121 T 379 75455 SENSE 0.2860.281 / 0.314 0.001 S 0.221 0.006 S 0.396 0.095 T 372 75463 SENSE 0.6010.011 S 0.141 0.221 / 0.163 0.049 S 1.252 0.058 T 394 75543 SENSE 0.3960.042 S 0.136 0.054 T 0.088 0.192 T 1.426 0.078 T S: represents thetransgenic plants showed statistically significant trait improvement ascompared to the reference (p < 0.05) T: represents the transgenic plantsshowed a trend of trait improvement as compared to the reference with p< 0.2 /: represents the transgenic plants didn't show any alteration orhad unfavorable change in traits examined compared to the reference inthe current dataset

Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination andsubsequent seedling growth, one being the availability of water. Genes,which can directly affect the success rate of germination and earlyseedling growth, are potentially useful agronomic traits for improvingthe germination and growth of crop plants under drought stress. Thisscreen identified genes for recombinant DNA that imparts enhance osmoticstress tolerance, a surrogate for enhanced water use efficiency, asshown in Arabidopsis seed when PEG was used to induce osmotic stress ongerminating transgenic lines of seeds.

T2 seeds were plated on BASTA selection plates containing 3% PEG andgrown under standard light and temperature conditions. Seeds were platedon each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10 μg/mlglufosinate. Plates were placed at 4° C. for 3 days to stratify seeds.On day 11, plants were measured for primary root length. After 3 moredays of growth, i.e. at day 14, plants were scored for transgenicstatus, primary root length, growth stage, visual color, and theseedlings were pooled for fresh weight measurement. A photograph of thewhole plate was taken on day 14.

Seedling weight and root length were analyzed as quantitative responsesaccording to example 1M. The final growth stage at day 14 was scored assuccess or failure based on whether the plants reached 3 rosette leavesand size of leaves are greater than 1 mm. The growth stage data wasanalyzed as a qualitative response according to example 1L. Table 7provides a list of recombinant DNA constructs that improve osmoticstress tolerance in transgenic plants.

TABLE 7 Seedling Weight Root Length Root Length Pep at day 14 at day 11at day 14 Growth Stage SEQ Orien- p- p- p- RS p- ID Gene tation deltavalue c delta value c delta value c mean value c 275 10180 ANTI- 0.3220.024 S 0.154 0.112 T 0.077 0.233 / 2.679 0.014 S SENSE 210 12919 SENSE0.523 0.001 S 0.15 0.104 T 0.134 0.211 / 1.067 0.084 T 218 16004 ANTI-0.58 0.005 S 0.109 0.465 / 0.137 0.048 S 0.96 0.183 T SENSE 243 19705SENSE 0.415 0.098 T 0.225 0.014 S 0.185 0.104 T 2.87 0.009 S 244 19737SENSE 0.71 0.065 T 0.163 0.454 / 0.127 0.477 / 2.147 0.036 S 238 19757SENSE 0.876 0.006 S 0.095 0.477 / 0.173 0.016 S 2.298 0.019 S 236 19902SENSE 0.635 0.012 S 0.233 0.044 S 0.1 0.104 T 2.314 0.016 S 230 19964SENSE 0.421 0.002 S 0.313 0.027 S 0.388 0.004 S 2.051 0.038 S 247 71330SENSE 0.35 0.003 S −0.09 0.453 / 0.137 0.189 T 3.292 0.003 S 220 71546SENSE 0.892 0.003 S 0.32 0.005 S 0.266 0.028 S 3.207 0.005 S 310 73480SENSE 0.257 0.053 T 0.12 0.058 T 0.069 0.487 / 3.231 0.004 S 254 73651SENSE 0.581 0.04 S 0.053 0.73 / 0.034 0.609 / 2.992 0.004 S 252 73685SENSE 0.467 0.024 S 0.137 0.171 T 0.006 0.933 / 1.556 0.026 S 262 73769SENSE 0.532 0.005 S 0.243 0.073 T 0.043 0.7 / 2.939 0.02 S 284 74340SENSE 0.56 0.003 S 0.258 0.049 S 0.237 0.036 S 3.541 0 S 293 74374 SENSE0.587 0.081 T 0.361 0.011 S 0.251 0.009 S 3.001 0.003 S 336 74504 SENSE0.801 0.003 S 0.185 0.021 S 0.111 0.021 S 4 0 S 337 74528 SENSE 0.5520.026 S 0.107 0.338 / −0.112 0.418 / 4 0 S 341 74554 SENSE 0.726 0.001 S0.337 0.012 S 0.224 0.065 T 3.462 0.001 S 343 74590 SENSE 0.511 0.034 S0.067 0.671 / 0.052 0.753 / 2.479 0.029 S 289 74608 SENSE 0.448 0.043 S0.175 0.298 / 0.241 0.109 T 2.617 0.016 S 300 74670 SENSE 0.855 0 S0.154 0.059 T −0.063 0.447 / 2.331 0.013 S 355 74951 SENSE 0.558 0.009 S0.488 0.003 S 0.517 0.001 S 3.302 0.003 S 363 74993 SENSE 0.677 0 S0.147 0 S 0.028 0.499 / 3.211 0.005 S 376 75418 SENSE 0.339 0.06 T 0.0230.763 / 0.263 0.016 S 0.811 0.246 / 381 75456 SENSE 0.43 0.044 S 0.230.03 S 0.198 0.003 S 2.674 0.004 S 383 75492 SENSE 0.434 0.018 S 0.0730.024 S 0.09 0.093 T 0.955 0.186 T 402 75536 SENSE 0.214 0.066 T 0.1460.015 S 0.221 0.025 S −1.33 0.996 / 389 75564 SENSE 0.407 0.043 S 0.0530.623 / 0.074 0.569 / 1.839 0.058 T 395 75567 SENSE 0.44 0.02 S −0.0770.522 / −0.107 0.291 / 2.4 0.034 S 393 75590 SENSE 0.431 0.006 S 0.210.022 S 0.195 0.014 S 2.848 0.006 S S: represents the transgenic plantsshowed statistically significant trait improvement as compared to thereference (p < 0.05) T: represents the transgenic plants showed a trendof trait improvement compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined compared to the reference in thecurrent dataset

Cold Shock Tolerance Screen

This screen identified genes for recombinant DNA that imparts enhancedsold tolerance as shown in Arabidopsis plants transformed with the genesof interest that are more tolerant to cold stress subjected during day 8to day 28 after seed planting. During these crucial early stages,seedling growth and leaf area increase were measured to assess tolerancewhen Arabidopsis seedlings were exposed to low temperatures. Using thisscreen, genetic alterations can be found that enable plants to germinateand grow better than wild type plants under sudden exposure to lowtemperatures.

Eleven seedlings from T2 seeds of each transgenic line plus one controlline were plated together on a plate containing ½× Gamborg Salts with0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then orientedhorizontally and stratified for three days at 4° C. At day three, plateswere removed from stratification and exposed to standard conditions (16hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At dayeight, plates were removed from standard conditions and exposed to coldshock conditions (24 hr photoperiod, 8° C. at both day and night) untilthe final day of the assay, i.e. day 28. Rosette areas were measured atday 8 and day 28, which were analyzed as quantitative responsesaccording to example 1M. Table 8 provides a list of recombinantnucleotides that improve cold shock stress tolerance in plants.

TABLE 8 difference in rosette rosette area rosette area area between day28 Pep at day 8 at day 28 and day 8 SEQ Con- Orien- p- p- p- IDstruct_id tation Delta value c delta value c delta value c 209 13222SENSE −0.562 0.978 / 0.396 0.074 T 0.581 0.058 T 216 14837 SENSE −0.080.661 / 1.007 0.002 S 1.154 0.002 S 212 14841 SENSE 0.662 0.023 S 1.0260.001 S 1.088 0.004 S 231 19814 SENSE 0.38 0.008 S 0.328 0.027 S 0.550.01 S 234 71072 SENSE −0.054 0.58 / 0.736 0.005 S 0.662 0.027 S 22471148 SENSE −0.43 0.899 / 0.189 0.14 T 0.643 0.02 S 217 71253 SENSE0.318 0.004 S 0.442 0.019 S 0.54 0.009 S 251 71689 SENSE −0.566 0.907 /0.62 0.01 S 0.879 0.043 S 312 73513 SENSE 0.48 0.009 S 0.61 0.022 S0.439 0.074 T 314 73527 SENSE 0.499 0.034 S 0.9 0.001 S 1.087 0.001 S321 74115 SENSE 0.254 0.17 T 0.896 0.002 S 1.029 0.002 S 324 74165 SENSE0.142 0.28 / 0.293 0.115 T 0.61 0.018 S 286 74345 SENSE −0.109 0.746 /0.819 0.004 S 1.148 0.009 S 333 74418 SENSE 0.421 0.002 S 1.012 0.003 S0.565 0.038 S 337 74528 SENSE 0.395 0.004 S 0.544 0.018 S 0.725 0.009 S294 74618 SENSE 0.274 0.003 S 0.781 0.011 S 0.941 0.033 S 305 74685SENSE −0.238 0.923 / 0.758 0.001 S 1.076 0 S 360 74919 SENSE 0.221 0.214/ 0.358 0.053 T 0.388 0.108 T 356 74940 SENSE 0.28 0.143 T 0.903 0.001 S0.952 0.002 S 358 74954 SENSE 0.197 0.176 T 0.602 0.013 S 0.712 0.024 S364 75337 SENSE 0.669 0.002 S 0.695 0.002 S 0.603 0.014 S 381 75456SENSE 0.413 0.034 S 0.895 0.002 S 0.998 0 S 380 75491 SENSE 0.324 0.047S 0.914 0.001 S 1.11 0.001 S S: represents the transgenic plants showedstatistically significant trait improvement as compared to the reference(p < 0.05) T: represents the transgenic plants showed a trend of traitimprovement compared to the reference with p < 0.2 /: represents thetransgenic plants didn't show any alteration or had unfavorable changein traits examined compared to the reference in the current dataset.

Cold Germination Tolerance Screen

This screen identified genes for recombinant DNA that imparts enhancedcold tolerance as shown in Arabidopsis plants transformed with the genesof interests are resistant to cold stress based on their rate ofdevelopment, root growth and chlorophyll accumulation under lowtemperature conditions.

T2 seeds were plated and all seedlings used in the experiment were grownat 8° C. Seeds were first surface disinfested using chlorine gas andthen seeded on assay plates containing an aqueous solution of ½×Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo.,USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/mlglufosinate with the final pH adjusted to 5.8 using KOH. Test plateswere held vertically for 28 days at a constant temperature of 8° C., aphotoperiod of 16 hr, and average light intensity of approximately 100umol/m²/s. At 28 days post plating, root length was measured, growthstage was observed, the visual color was assessed, and a whole platephotograph was taken.

The root length at day 28 was analyzed as a quantitative responseaccording to example 1M. The growth stage at day 7 was analyzed as aqualitative response according to example 1L. Table 9 provides a list ofrecombinant DNA constructs that improve cold stress tolerance intransgenic plants.

TABLE 9 Root Length Growth Stage Pep at day 28 at day 28 SEQ Con- Orien-p- RS p- ID struct_id tation delta value c mean value c 269 10814 ANTI-0.218 0.009 S 3.148 0.007 S SENSE 208 17465 SENSE 0.269 0.027 S 3.2460.004 S 223 17919 SENSE 0.366 0.002 S 4 0 S 346 18020 SENSE 0.112 0.105T 3.016 0.014 S 406 18026 SENSE 0.407 0.002 S 2.111 0.039 S 226 18844SENSE 0.464 0.012 S 2.927 0.005 S 225 19647 SENSE 0.479 0.002 S 3.1290.008 S 232 19720 SENSE 0.295 0.003 S 4 0 S 229 19750 SENSE 0.224 0.006S 3.161 0.006 S 245 19812 SENSE 0.261 0.002 S 3.022 0.014 S 235 19949SENSE 0.226 0 S 3.093 0.01 S 220 71546 SENSE 0.3 0.001 S 3.181 0.006 S274 73061 SENSE 0.306 0.002 S 3.447 0.001 S 256 73271 SENSE 0.062 0.209/ 4 0 S 257 73282 SENSE 0.216 0.023 S 4 0 S 255 73342 SENSE 0.366 0 S 40 S 292 74383 SENSE 0.182 0.019 S 4 0 S 336 74504 SENSE 0.368 0.001 S 40 S 337 74528 SENSE 0.202 0 S 4 0 S 339 74541 SENSE 0.338 0.002 S 4 0 S341 74554 SENSE 0.239 0.001 S 4 0 S 342 74578 SENSE 0.53 0 S 4 0 S 28974608 SENSE 0.261 0.003 S 4 0 S 351 74880 SENSE 0.21 0.04 S 2.977 0.017S 359 74907 SENSE 0.253 0.004 S 1.951 0.096 T 372 75463 SENSE 0.4670.002 S 4 0 S 373 75475 SENSE 0.307 0.009 S 3.496 0 S 382 75480 SENSE0.268 0.012 S 2.955 0.018 S 392 75554 SENSE 0.149 0.039 S 4 0 S 39575567 SENSE 0.236 0.003 S 4 0 S 347 75850 SENSE 0.616 0 S 3.053 0.002 S348 75861 SENSE 0.272 0.001 S 4 0 S 349 75875 SENSE 0.138 0.066 T 4 0 S403 75991 SENSE 0.038 0.296 / 2.667 0.051 T S: represents the transgenicplants showed statistically significant trait improvement as compared tothe reference (p < 0.05) T: represents the transgenic plants showed atrend of trait improvement as compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined compared to the reference in thecurrent dataset

Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade thatincludes the elongation of the petiole, a change in the leaf angle, anda reduction in chlorophyll content. While these changes may confer acompetitive advantage to individuals, in a monoculture the shadeavoidance response is thought to reduce the overall biomass of thepopulation. Thus, genetic alterations that prevent the shade avoidanceresponse may be associated with higher yields. Genes that favor growthunder low light conditions may also promote yield, as inadequate lightlevels frequently limit yield. This screen identified genes forrecombinant DNA that imparts enhanced shade tolerance in Arabidopsisplants that show an attenuated shade avoidance response and/or growbetter than control plants under low light intensity. Of particularinterest, we were looking for plants that didn't extend their petiolelength, had an increase in seedling weight relative to the reference andhad leaves that were more close to parallel with the plate surface.

T2 seeds were plated on glufosinate selection plates with ½ MS medium.Seeds were sown on ½×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants weregrown on vertical plates at a temperature of 22° C. at day, 20° C. atnight and under low light (approximately 30 uE/m²/s, far/red ratio(655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680).Twenty-three days after seedlings were sown, measurements were recordedincluding seedling status, number of rosette leaves, status of flowerbud, petiole leaf angle, petiole length, and pooled fresh weights. Adigital image of the whole plate was taken on the measurement day.Seedling weight and petiole length were analyzed as quantitativeresponses according to example 1M. The number of rosette leaves,flowering bud formation and leaf angel were analyzed as qualitativeresponses according to example 1L.

Table 10 provides a list of recombinant DNA constructs that improveshade tolerance in plants

TABLE 10 Petiole length seedling weight Leaf angle Number of rosette Pepat day 23 day 23 at day 23 leaves at day 23 SEQ Con- Orien- p- p- RS p-RS p- ID struct_id tation delta value c delta value c mean value c meanvalue c 407 11810 ANTI- −0.138 0.03 S 0.158 0.245 / 0.246 0.296 / −0.10.822 / SENSE 267 16014 SENSE −0.267 0.012 S −1.129 0.015 / 0.042 0.425/ 0.773 0.209 / 249 71571 SENSE −0.548 0.005 S −0.212 0.177 / 0.3250.224 / 0.611 0.22 / 273 72984 SENSE −0.219 0.064 T −0.126 0.178 / 0.120.315 / 0.476 0.28 / 280 73044 SENSE −0.722 0.083 T −0.572 0.173 /−0.032 1 / −1.35 0.992 / 307 73476 SENSE −0.01 0.905 / 0.103 0.354 /0.739 0.177 T 1.896 0.053 T 311 73482 SENSE −0.394 0.107 T −0.095 0.611/ 0.045 0.375 / 0.252 0.376 / 306 73487 SENSE 0.064 0.457 / −0.131 0.583/ 1.656 0.051 T 1.131 0.15 T 261 74306 SENSE −1.107 0.13 T −1.924 0.116/ 0.291 0.261 / −0.103 0.591 / 282 74327 SENSE 0.029 0.777 / 0.105 0.762/ 0.666 0.12 T 1.252 0.116 T 330 74476 SENSE −0.092 0.072 T −0.613 0.107/ 0.271 0.246 / −0.026 0.511 / S: represents the transgenic plantsshowed statistically significant trait improvement as compared to thereference (p < 0.05) T: represents the transgenic plants showed a trendof trait improvement as compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined compared to the reference in thecurrent dataset.

Early Plant Growth and Development Screen

This screen identified genes for recombinant DNA that imparts enhancedearly plant growth and development, a surrogate for increased yield, asshown in Arabidopsis plants examined in a plate based phenotypicanalysis platform for the rapid detection of phenotypes that are evidentduring the first two weeks of growth. In this screen, we were lookingfor genes that confer advantages in the processes of germination,seedling vigor, root growth and root morphology under non-stressedgrowth conditions to plants. The transgenic plants with advantages inseedling growth and development were determined by the seedling weightand root length at day 14 after seed planting.

T2 seeds were plated on glufosinate selection plates and grown understandard conditions (˜100 uE/m²/s, 16 h photoperiod, 22° C. at day, 20°C. at night). Seeds were stratified for 3 days at 4° C. Seedlings weregrown vertically (at a temperature of 22° C. at day 20° C. at night).Observations were taken on day 10 and day 14. Both seedling weight androot length at day 14 were analyzed as quantitative responses accordingto example 1M.

Table 11 provides a list recombinant DNA constructs that improve earlyplant growth and development.

TABLE 11 Root Length Root Length Pep at day 10 at day 14 Seedling WeightSEQ Con- Orien- p- p- p- ID struct_id tation delta value c delta value cdelta value c 213 13478 ANTI- 0.242 0.062 T 0.159 0.075 T 0.603 0.013 SSENSE 227 19525 SENSE 0.249 0.183 T 0.245 0.001 S 0.385 0.062 T 22170109 SENSE 0.282 0.001 S 0.23 0.001 S 0.564 0 S 248 71332 SENSE 0.0710.548 / 0.093 0.17 T 0.419 0.009 S 220 71546 SENSE 0.307 0.057 T 0.2310.013 S 0.561 0.006 S 246 71556 SENSE 0.408 0.008 S 0.291 0.011 S 0.3280.263 / 408 72418 SENSE 0.144 0.063 T 0.17 0 S 0.46 0.006 S 253 72636SENSE 0.486 0.026 S 0.339 0 S 0.705 0.006 S 279 72957 SENSE 0.228 0.053T 0.158 0.089 T 0.371 0.08 T 309 73418 SENSE 0.242 0.082 T 0.148 0.089 T0.212 0.085 T 316 73530 SENSE 0.239 0.023 S 0.17 0.003 S 0.442 0.001 S313 73550 SENSE 0.181 0.046 S 0.113 0.016 S 0.304 0.045 S 259 73913SENSE 0.243 0.069 T 0.091 0.26 / 0.452 0.015 S 325 74106 SENSE 0.2570.003 S 0.192 0.015 S 0.34 0.052 T 318 74125 SENSE 0.104 0.549 / 0.1390.048 S 0.524 0.002 S 320 74126 SENSE 0.149 0.082 T 0.065 0.284 / 0.3540.127 T 322 74127 SENSE 0.381 0 S 0.212 0.006 S 0.63 0.002 S 323 74128SENSE 0.213 0.091 T 0.12 0.161 T 0.541 0.002 S 326 74130 SENSE 0.140.002 S 0.144 0.001 S 0.003 0.955 / 327 74132 SENSE 0.134 0.275 / 0.1440.049 S 0.336 0.025 S 328 74144 SENSE 0.137 0.217 / 0.134 0.065 T 0.4920.012 S 319 74161 SENSE 0.205 0.02 S 0.131 0.018 S 0.459 0.035 S 26374237 SENSE 0.253 0 S 0.189 0.002 S 0.408 0.013 S 265 74256 SENSE 0.280.04 S 0.202 0.012 S 0.461 0.034 S 260 74305 SENSE 0.184 0.063 T 0.1310.026 S 0.257 0.121 T 281 74323 SENSE 0.13 0.08 T 0.067 0.054 T 0.4260.005 S 285 74341 SENSE 0.185 0.044 S 0.07 0.106 T 0.144 0.191 T 29374374 SENSE 0.103 0.187 T 0.171 0.008 S 0.518 0.023 S 302 74385 SENSE0.051 0.461 / −0.043 0.693 / 0.214 0.098 T 303 74386 SENSE 0.211 0.12 T0.136 0.092 T 0.081 0.616 / 304 74387 SENSE 0.11 0.528 / 0.17 0.052 T0.378 0.027 S 350 74548 SENSE 0.169 0.041 S 0.09 0.046 S 0.336 0.08 T288 74604 SENSE 0.157 0.231 / 0.181 0.014 S 0.586 0.001 S 290 74609SENSE 0.24 0.029 S 0.102 0.227 / 0.457 0.014 S 287 74616 SENSE 0.1920.047 S 0.137 0.125 T 0.286 0.204 / 295 74619 SENSE 0.181 0.415 / 0.1530.149 T 0.448 0.056 T 296 74622 SENSE 0.083 0.481 / 0.071 0.331 / 0.3110.076 T 298 74631 SENSE 0.203 0.02 S 0.115 0.019 S −0.049 0.633 / 35374915 SENSE 0.202 0.004 S 0.136 0.027 S 0.409 0.012 S 354 74927 SENSE0.103 0.148 T 0.117 0.021 S 0.319 0.056 T 356 74940 SENSE 0.127 0.001 S0.117 0.049 S 0.344 0.028 S 371 75312 SENSE 0.191 0.055 T 0.155 0.006 S0.526 0.001 S 365 75339 SENSE 0.168 0.029 S 0.031 0.704 / 0.21 0.208 /374 75440 SENSE 0.15 0.037 S 0.059 0.566 / −0.039 0.923 / 372 75463SENSE 0.392 0.006 S 0.284 0.011 S 0.432 0.15 T 375 75488 SENSE 0.1010.289 / 0.083 0.113 T 0.444 0.003 S S: represents the transgenic plantsshowed statistically significant trait improvement as compared to thereference (p < 0.05) T: represents the transgenic plants showed a trendof trait improvement as compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined compared to the reference in thecurrent dataset

Late Plant Growth and Development Screen

This screen identified genes for recombinant DNA that imparts enhancedlate plant growth and development, a surrogate for increased yield, asshown in Arabidopsis plants examined in a soil based phenotypic platformto identify genes that confer advantages in the processes of leafdevelopment, flowering production and seed maturity to plants.

Arabidopsis plants were grown on a commercial potting mixture (Metro Mix360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium gradehorticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processedbark ash, 1-15% pine bark and a starter nutrient charge. Soil wassupplemented with Osmocote time-release fertilizer at a rate of 30mg/ft³. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C.and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots wereordered in a 4 by 8 grid in standard greenhouse flat. Plants were grownin environmentally controlled rooms under a 16 h day length with anaverage light intensity of ˜200 μmoles/m²/s. Day and night temperatureset points were 22° C. and 20° C., respectively. Humidity was maintainedat 65%. Plants were watered by sub-irrigation every two days on averageuntil mid-flowering, at which point the plants were watered daily untilflowering was complete.

Application of the herbicide glufosinate was performed to select T2individuals containing the target transgene. A single application ofglufosinate was applied when the first true leaves were visible. Eachpot was thinned to leave a single glufosinate-resistant seedling ˜3 daysafter the selection was applied.

The rosette radius was measured at day 25. The silique length wasmeasured at day 40. The plant parts were harvested at day 49 for dryweight measurements if flowering production was stopped. Otherwise, thedry weights of rosette and silique were carried out at day 53. The seedswere harvested at day 58. All measurements were analyzed as quantitativeresponses according to example 1M.

Table 12 provides a list of recombinant DNA constructs that improve lateplant growth and development.

TABLE 12 Pep Con- Rosette Dry Weight Rosette Radius Seed Dry WeightSilique Dry Weight Silique Length SEQ struct p- p- p- p- p- ID id deltavalue c delta value c Delta value c delta value c delta value c 21313478 0.054 0.365 / 0.217 0.013 S 0.052 0.347 / 0.293 0.084 T 0.0790.021 S 241 19787 0.233 0.052 T 0.067 0.022 S 0.66 0.019 S −0.02 0.75 /0.06 0.006 S 268 72751 0.287 0.012 S −0.141 0.714 / −0.991 0.973 / 0.0940.099 T −0.024 0.675 / S: represents the transgenic plants showedstatistically significant trait improvement as compared to the reference(p < 0.05) T: represents the transgenic plants showed a trend of traitimprovement compared to the reference with p < 0.2 /: represents thetransgenic plants didn't show any alteration or had unfavorable changein traits examined compared to the reference in the current dataset

Limited Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chloroticand have less biomass. This screen identified genes for recombinant DNAthat imparts enhanced nitrogen use efficiency as shown in Arabidopsisplants transformed with the gene of interest that are altered in theirability to accumulate biomass and/or retain chlorophyll under lownitrogen condition.

T2 seeds were plated on glufosinate selection plates containing 0.5×N-Free Hoagland's T 0.1 mM NH₄NO₃ T 0.1% sucrose T 1% phytagel media andgrown under standard light and temperature conditions. At 12 days ofgrowth, plants were scored for seedling status (i.e. viable ornon-viable) and root length. After 21 days of growth, plants were scoredfor BASTA resistance, visual color, seedling weight, number of greenleaves, number of rosette leaves, root length and formation of floweringbuds. A photograph of each plant was also taken at this time point.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The number green leaves, the numberof rosette leaves and the flowerbud formation were analyzed asqualitative responses according to example 1L. The leaf color raw datawere collected on each plant as the percentages of five color elements(Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using acomputer imaging system. A statistical logistic regression model wasdeveloped to predict an overall value based on five colors for eachplant.

Table 13 provides a list of recombinant DNA constructs that improve lownitrogen availability tolerance in plants.

TABLE 13 Number of green Pep Con- leaves leaf color Root Length RosetteWeight SEQ struct RS p- p- RS p- p- ID id mean value c delta value cmean value c delta value c 334 10150 1.496 0.007 S 0.974 0.007 S −0.2850.022 / −0.024 0.572 / 222 10335 0.993 0.014 S 0.953 0.01 S −0.359 0 /−0.057 0.122 / 277 11145 0.368 0.41 / 1.059 0.004 S −0.141 0.211 /−0.086 0.143 / 361 11735 0.522 0.554 / 1.132 0.003 S −0.134 0.147 /0.082 0.001 S 206 12030 0.57 0.321 / 0.944 0.027 S −0.28 0.005 / −0.0310.545 / 368 12189 0.134 0.791 / 0.76 0.034 S −0.147 0.024 / −0.082 0.099/ 308 73465 0.591 0.195 T 0.586 0.061 T −0.03 0.631 / −0.04 0.249 / 32874144 0.845 0.069 T 1.021 0.003 S 0.181 0.04 S 0.028 0.481 / 331 744171.051 0.021 S 1.4 0.001 S −0.155 0.019 / −0.071 0.16 / 338 74588 0.4840.31 / 1.059 0.01 S −0.101 0.167 / −0.051 0.608 / 369 75321 0.607 0.041S 0.317 0.423 / −0.095 0.238 / 0.056 0.021 S 377 75419 −0.779 0.198 /0.635 0.019 S −0.116 0.024 / 0.009 0.795 / 385 75424 −0.354 0.537 /1.489 0 S −0.245 0.003 / −0.062 0.165 / 391 75506 0.969 0 S 0.588 0.049S 0.081 0.108 T −0.026 0.545 / 388 75528 −0.026 0.932 / 0.229 0.594 /−0.042 0.534 / 0.111 0.058 T 396 75544 0.197 0.369 / 1.033 0.006 S−0.283 0.034 / −0.134 0.021 / 398 75546 0.577 0.001 S 1.108 0.002 S−0.321 0.006 / 0.01 0.765 / 390 75553 −0.182 0.643 / 0.425 0.058 T−0.104 0.12 / −0.096 0.104 / 397 75556 0.766 0.01 S −0.544 0.179 / 0 1 /0.271 0 S 399 75558 0.343 0.406 / 0.861 0.008 S −0.102 0.035 / −0.1060.003 / 387 75575 0.087 0.83 / 1.147 0.001 S −0.117 0.093 / −0.03 0.325/ 401 75583 −0.621 0.399 / −0.166 0.595 / −0.01 0.906 / 0.162 0.002 S344 75834 0.425 0.038 S 0.589 0.03 S −0.067 0.343 / 0.028 0.429 / 34575835 0.486 0.266 / 0.228 0.499 / −0.16 0.026 / 0.159 0 S S: representsthe transgenic plants showed statistically significant trait improvementas compared to the reference (p < 0.05) T: represents the transgenicplants showed a trend of trait improvement compared than the referencewith p < 0.2 /: represents the transgenic plants didn't show anyalteration or had unfavorable change in traits examined compared to thereference in the current dataset

Statistic Analysis for Qualitative Responses

Table 14 provides a list of responses that were analyzed as qualitativeresponses

TABLE 14 response screen categories (success vs. failure) wiltingresponse Risk Soil drought tolerance screen non-wilted vs. wilted Scoregrowth stage at day 14 heat stress tolerance screen 50% of plants reachstage 1.03 vs. not growth stage at day 14 salt stress tolerance screen50% of plants reach stage 1.03 vs. not growth stage at day 14 PEGinduced osmotic stress tolerance 50% of plants reach stage 1.03 vs. notscreen growth stage at day 7 cold germination tolerance screen 50% ofplants reach stage 0.5 vs. not number of rosette leaves Shade tolerancescreen 5 leaves appeared vs. not at day 23 flower bud formation at Shadetolerance screen flower buds appear vs. not day 23 leaf angle at day 23Shade tolerance screen >60 degree vs. <60 degree number of green leavesat limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not day21 number of rosette leaves limited nitrogen tolerance screen 6 or 7leaves appeared vs. not at day 21 Flower bud formation at limitednitrogen tolerance screen flower buds appear vs. not day 21

Plants were grouped into transgenic and reference groups and were scoredas success or failure according to Table 16. First, the risk (R) wascalculated, which is the proportion of plants that were scored as offailure plants within the group. Then the relative risk (RR) wascalculated as the ratio of R (transgenic) to R (reference). Risk score(RS) was calculated as −log₂ ^(RR). Subsequently the risk scores frommultiple events for each transgene of interest were evaluated forstatistical significance by t-test using S-PLUS statistical software(S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). RSwith a value greater than 0 indicates that the transgenic plants performbetter than the reference. RS with a value less than 0 indicates thatthe transgenic plants perform worse than the reference. The RS with avalue equal to 0 indicates that the performance of the transgenic plantsand the reference don't show any difference.

Statistic Analysis for Quantitative Responses

Table 15 provides a list of responses that were analyzed as quantitativeresponses.

TABLE 15 response screen seed yield Soil drought stress tolerance screenseedling weight at day 14 heat stress tolerance screen root length atday 14 heat stress tolerance screen seedling weight at day 14 saltstress tolerance screen root length at day 14 salt stress tolerancescreen root length at day 11 salt stress tolerance screen seedlingweight at day 14 PEG induced osmotic stress tolerance screen root lengthat day 11 PEG induced osmotic stress tolerance screen root length at day14 PEG induced osmotic stress tolerance screen rosette area at day 8cold shock tolerance screen rosette area at day 28 cold shock tolerancescreen difference in rosette area cold shock tolerance screen from day 8to day 28 root length at day 28 cold germination tolerance screenseedling weight at day 23 Shade tolerance screen petiole length at day23 Shade tolerance screen root length at day 14 Early plant growth anddevelopment screen Seedling weight at day 14 Early plant growth anddevelopment screen Rosette dry weight at day 53 Late plant growth anddevelopment screen rosette radius at day 25 Late plant growth anddevelopment screen seed dry weight at day 58 Late plant growth anddevelopment screen silique dry weight at day 53 Late plant growth anddevelopment screen silique length at day 40 Late plant growth anddevelopment screen Seedling weight at day 21 Limited nitrogen tolerancescreen Root length at day 21 Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂ calculation.The Delta was calculated as log₂M(transgenic)-log₂M(reference).Subsequently the mean delta from multiple events of the transgene ofinterest was evaluated for statistical significance by t-test usingS-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful,Seattle, Wash., USA). The Delta with a value greater than 0 indicatesthat the transgenic plants perform better than the reference. The Deltawith a value less than 0 indicates that the transgenic plants performworse than the reference. The Delta with a value equal to 0 indicatesthat the performance of the transgenic plants and the reference don'tshow any difference.

Example 2

This example illustrates the identification of homologs of the cognateproteins of the genes identified as imparting an enhanced trait.

A BLAST searchable “All Protein Database” was constructed of knownprotein sequences using a proprietary sequence database and the NationalCenter for Biotechnology Information (NCBI) non-redundant amino aciddatabase (nr.aa). For each organism from which a DNA sequence providedherein was obtained, an “Organism Protein Database” was constructed ofknown protein sequences of the organism; the Organism Protein Databaseis a subset of the All Protein Database based on the NCBI taxonomy IDfor the organism.

The All Protein Database was queried using amino acid sequence ofcognate protein for gene DNA used in trait-improving recombinant DNA,i.e. sequences of SEQ ID NO: 205 through SEQ ID NO: 408 using “blastp”with E-value cutoff of 1e-8. Up to 1000 top hits were kept, andseparated by organism names. For each organism other than that of thequery sequence, a list was kept for hits from the query organism itselfwith a more significant E-value than the best hit of the organism. Thelist contains likely duplicated genes, and is referred to as the CoreList. Another list was kept for all the hits from each organism, sortedby E-value, and referred to as the Hit List.

The Organism Protein Database was queried using amino acid sequences ofSEQ ID NO: 205 through SEQ ID NO: 408 using “blastp” with E-value cutoffof 1e-4. Up to 1000 top hits were kept. A BLAST searchable database wasconstructed based on these hits, and is referred to as “SubDB”. SubDBwas queried with each sequence in the Hit List using “blastp” withE-value cutoff of 1e-8. The hit with the best E-value was compared withthe Core List from the corresponding organism. The hit is deemed alikely ortholog if it belongs to the Core List, otherwise it is deemednot a likely ortholog and there is no further search of sequences in theHit List for the same organism. Likely orthologs from a large number ofdistinct organisms were identified and are reported by amino acidsequences of SEQ ID NO: 409 to SEQ ID NO: 19247. These orthologs arereported in Tables 2 as homologs to the proteins cognate to genes usedin trait-improving recombinant DNA.

TABLE 2 SEQ ID NO: homolog SEQ ID NOs 206: 9461 19072 14856 5315 772611678 8466 4567 9541 4039 2736 826 4434 18755 7163 19021 16621 18594207: 11732 5088 14017 14658 8659 17828 14150 11838 7420 577 13978 30899378 8177 5473 4151 15455 3299 3397 17568 12473 2946 17428 3126 154083997 18806 10067 3246 19105 208: 11715 12490 18003 15562 5946 18565 555013362 1866 4207 6966 6785 16958 17740 12495 9774 3632 7059 937 1064312198 6865 13806 1023 12708 14552 16746 18201 16712 7735 209: 3105 154181368 15360 5654 7902 3038 16108 14416 16444 10078 17851 12848 1518312997 18090 18617 1312 16630 4080 4082 7689 19205 17262 13305 5780 1054414798 16363 16166 17737 210: 17539 13894 15187 11374 17968 9384 764513991 5852 211: 16101 13052 9940 10298 9899 15964 10104 18651 6853 126191551 4764 684 13219 15864 9975 3261 4237 2710 18739 15477 7248 582 15025212: 11577 10930 12130 13904 13619 10462 9686 10796 9854 12277 147084822 437 6953 7206 9936 2948 7987 13119 2409 213: 11281 4744 9418 86073777 4299 2607 2319 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18010 14502 4220 6076 1110812418 16973 8611 17786 18551 11710 1128 4641 1538 16850 15885 4965 151506585 18750 6697 10746 11219 12375 13129 559 6520 4368 2832 3991 135433423 2624 18457 10864 11199 16932 16270 5072 17123 5876 14612 896 1515616852 18175 10945 18855 16525 1748 5964 11300 8621 7611 9963 9241 66075035 14105 1285 11382 13286 12956 17420 6377 1873 17080 19067 9405 872913064 2473 14203 6583 10484 11942 9591 5237 2321 3769 3885 7954 1799714024 5941 9954 18484 18027 18467 18028 17973 17972 17970 17969 156305302 6762 16042 17109 8202 9781 3057 12195 14637 2924 18035 5223 15409595 5651 5083 17949 19109 11395 6580 10789 13695 15802 5389 18245 185597398 13922 18325 18513 18472 328 327 7707 7353 7349 7351 7348 7218 72147201 7224 7220 7200 2490 3607 15058 19237 15045 15907 16396 16398 1591215057 15636 16403 15079 15075 9155 18489 330 363: 18274 18488 1800711117 10258 9446 3371 18022 329 360 359 7341 17634 11532 3678 17861 114111850 6626 1745 15264 16539 2664 8699 5767 11011 8036 7857 1343 68398733 11515 14442 16737 6301 658 5340 6383 3012 4309 4229 16075 2351 893212221 5354 6706 3213 3896 17512 15668 15120 4825 18765 7855 4601 109912531 10828 17127 1411 5701 15745 17917 4012 11414 8414 1948 325 88823926 7772 18792 16498 13428 7045 7919 16773 3833 8372 5231 6402 88994814 12738 16127 326 12276 13535 12190 17785 10684 3455 14687 9274 1207715788 10337 9859 10204 9466 15723 7916 18347 6113 15339 18049 1416214158 7554 2228 5734 2183 1503 9273 13486 4699 18053 874 7758 7168 291116068 10075 5165 6610 8188 7505 5836 10110 3449 15698 17609 5486 101883795 3794 974 7376 8980 2807 2826 15414 3221 11000 16877 358 11643 1692814101 17431 14104 11922 14106 14118 14103 11917 14117 11877 9156 1552115519 15512 2076 7599 18286 16747 17450 10636 10883 19245 18626 27065629 17448 17445 13484 12611 12363 8462 8465 1521 11661 18323 1049518481 1858 7476 18811 19045 17901 1025 15261 14337 11009 802 1489 97515535 19225 4741 8379 9986 5615 9570 18249 1579 18874 14957 2833 1309711855 17444 2048 5863 16811 12374 13302 5440 14857 13516 10812 9419 677111768 12141 3543 6242 11404 18052 1963 2636 7150 2875 5500 15645 302419039 805 11538 2943 12915 17169 4471 17129 2420 9530 8029 15921 1604111094 10747 13795 2144 17511 12731 1983 6924 15147 18864 7598 2259 795018104 7123 13183 9498 651 5099 8353 13883 4389 2662 11940 19159 163644653 14754 12986 3439 9888 4998 8161 19192 17068 10691 934 606 769 846818297 1397 3167 10721 18909 18127 10323 820 8767 11231 10209 18039 47694271 914 5454 17761 1436 16749 12983 10781 7990 3461 11482 2488 260515929 19116 1132 11154 5499 16543 12081 4575 15011 17427 14182 114944324 686 8119 16545 1033 15056 15910 7850 13576 15542 16151 16155 1609513014 16069 3257 6401 17505 17504 17525 17524 2022 7377 432 16819 103322411 10331 2410 12098 12865 3223 4128 11048 1117 13770 13767 16781 1677716779 16801 17741 16774 4860 15510 12777 12778 14071 14067 14053 1406914091 14072 14075 8376 11001 9987 7853 5184 7393 7395 17909 3219 322017517 3203 17996 3205 17513 10899 3204 18474 18023 18329 600 6542 791116191 13238 15975 4924 9729 6091 4326 6820 13828 4812 9933 17784 164244768 15749 7309 10904 2071 4194 15043 2025 10355 5253 18478 13347 1850913344 18011 4578 8209 8302 10221 15948 8147 11081 18398 17612 16052 285315154 15669 4108 413 16888 8441 3479 1048 17549 9749 17291 14470 200813574 5988 3547 13320 12107 4540 6031 16301 6076 18372 10579 4109 1136619231 11108 8482 5412 12150 14436 18176 1610 674 6891 13730 8642 60953638 993 17479 11156 6014 4301 1954 10818 7039 15055 12418 10003 192413798 11179 15926 13552 9569 18113 9432 15846 14535 3145 12007 1613212934 1538 16847 15583 6741 5534 14502 4641 7188 15672 4220 11219 123757172 3983 2230 6072 5854 17508 17766 18490 15885 4617 4368 16850 139403991 1557 11615 2624 18508 18457 10864 18750 6697 1128 12487 18977 544511199 10746 16932 5601 16270 559 5072 16012 1609 13543 18010 5876 1855111710 16973 8611 17786 896 15150 6585 6520 13129 4965 18175 2832 109453423 17125 18855 16525 1748 5964 7611 14612 11300 17123 14105 1285 1515611382 12956 17420 6607 6377 1873 5035 9405 13286 8729 9963 8621 924113064 2473 14203 19067 17080 10484 16852 9591 6583 5237 3885 11942 140245941 2321 18484 18027 18467 18028 17973 17972 17970 17969 11616 530216042 6762 8202 17109 9781 3057 12195 18035 14637 2924 5223 595 56515083 15409 17949 19109 11395 13131 5389 6525 18579 18245 18559 158026580 13199 10789 12935 13695 14236 5044 6962 15953 11413 2116 18736 380313250 13906 7398 13922 11708 9202 18325 18513 6519 18472 327 328 79297353 7349 7351 7348 7218 7214 7201 7224 7220 7200 17337 7741 2490 68013458 13727 18735 316 321 333 322 324 320 319 323 6750 15058 19237 1504515907 16396 16398 15912 15057 15636 16403 15079 15075 18489 13580 7766364: 8684 16027 13149 10126 8454 15932 11869 13702 13698 7664 8628 30393267 365: 17649 8270 3136 10804 6247 8834 17636 15016 8936 2194 131574323 8741 11308 472 19056 366: 5012 2906 8001 12356 6089 18788 4785 61866359 6711 3320 11092 367: 7896 15587 5766 14289 18000 13369 8046 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8004 15938 15486 12018 6903 5465 5202 3639 10189 400017403 4727 18570 18571 18591 13103 7276 8327 8749 14147 13677 8146 101954684 11527 15892 11819 15958 3517 15190 16361 11510 369: 2084 15197 86628366 2930 6655 12876 8740 17571 13116 370: 1613 19204 11892 6151 1183416271 7629 371: 3593 6170 6205 17780 18378 1421 3053 15450 9886 1573918891 6294 10793 15529 13666 11730 18928 3004 6197 13102 16177 1392814868 12232 6033 7442 14783 15125 5741 864 13206 5210 12920 18629 1267116466 2407 5884 6651 14574 372: 15623 14801 953 10002 6238 10071 1375611624 16541 14110 3858 4119 4412 10719 373: 4316 13909 16449 17769 150064781 10038 14140 13489 16035 4824 9107 6225 16014 8175 17809 3179 1474016152 8688 16780 13019 17782 17245 374: 6174 6173 17263 5382 13230 41613835 10352 12036 2106 2087 12205 12076 13351 5940 7910 11809 1673015960 14680 8484 3972 4409 4808 18796 15783 12008 3864 440 18038 1924618731 16299 4005 5505 5331 12153 14304 12102 17171 375: 15552 2308 108214674 10575 4747 17033 5313 505 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15500 83391316 19032 6299 1170 8702 13733 10030 10478 10019 10089 4520 3432 1837718380 8333 8284 8313 2075 6604 16849 5563 10245 3095 10445 11875 696010489 4952 7044 8314 17763 8310 12788 8318 7964 8340 8343 8346 1962 88472058 16080 3127 15015 17613 17633 2348 8003 7373 7372 15767 12500 1606511929 8086 6387 14998 6461 11845 11762 11109 7190 17213 5075 5076 73677365 13195 7449 7491 7448 7535 11930 7290 7294 1927 1929 1975 1976 129278317 379: 789 2899 9238 3272 5648 3579 15295 6275 14031 16259 5495 109635221 2572 18153 15647 4771 8787 11837 4871 6699 10308 17936 16583 149637534 12772 9430 13248 6068 13071 380: 11132 17164 10120 13780 18790 39806453 16881 5663 9535 19104 1529 18961 2182 9230 8958 10766 3066 1451516060 4872 16197 5238 18266 381: 8677 2479 7211 7425 4341 4838 7350 72648013 7326 7209 7235 15830 5983 5979 5985 18656 7511 7198 15500 8339 6276299 16863 8702 15217 15219 15220 12265 2585 12272 12270 12268 8753 306111536 11534 4520 3432 18952 18377 18380 8333 8284 8313 2075 4839 66045557 3612 4952 7044 3124 13964 13963 7964 8343 8346 16080 7195 8003 737316065 8853 11929 7949 7962 6461 3480 11762 11845 17213 13195 7449 123487448 7446 11930 6644 17544 1979 1976 688 12002 692 12927 12924 129218317 382: 6843 6987 13364 15545 14423 4921 4285 2528 2557 18040 83509340 383: 15954 6049 18779 18043 2249 15965 384: 7925 14352 11545 1061516765 15479 8141 14076 15758 5411 12128 15947 3153 8503 16045 11187 36869965 1284 5392 16793 15607 5705 3059 7149 385: 12678 11761 19097 1508210667 4225 11019 8039 8375 17367 9578 18072 386: 10572 14694 5590 48763577 12056 17885 18628 17398 7894 7122 673 4475 387: 13459 5045 129992199 13901 17853 19179 15940 9881 12409 5036 14275 18503 8364 2168 80794928 2375 2053 17350 16572 10390 3007 7945 7762 641 388: 16871 976111846 15784 18262 18446 17041 2127 8909 17081 8129 10993 5337 2955 105501632 4698 18308 13085 15485 13327 16105 389: 2476 3590 2914 12550 125527501 14570 15819 11167 4325 19077 6830 4556 10406 12930 5172 5060 123893569 16656 5168 9775 14074 17762 9431 2976 16817 11358 11360 2041 1137510922 11355 11359 14965 10920 10918 3529 4796 11740 2289 15427 7180 92754129 7616 13151 18966 10689 14083 6228 2080 13696 5628 10009 411 1049915596 8605 17387 390: 13101 8214 18411 6355 17130 17131 17112 1710611037 5175 5207 16634 16638 18236 18250 13007 12625 10843 10358 4002 9057460 17283 10362 10388 10845 13303 2314 12875 17641 7106 16084 4137 94914494 14185 14189 14160 13722 10262 9561 10261 2652 16419 14234 3867 33944122 16608 17323 18139 16832 11596 16537 14193 14190 14231 14212 178327504 18708 7840 16675 16237 7108 13984 2646 18664 14232 8358 12918 44734497 431 13345 11107 14988 10858 8415 10522 18463 2013 10896 13582 14538762 16443 14346 11683 2065 10233 10846 10363 10391 10393 14422 993517273 17249 8363 10121 14942 391: 7780 7778 5749 16030 8501 14291 142886980 8600 7808 14360 14094 2039 3393 16652 5033 5164 14882 16247 424912196 17478 5851 448 19005 392: 9293 16557 723 6692 9622 2591 13628 69281592 393: 5309 16676 1992 5415 17277 4097 6941 16134 3966 10723 1227512413 9592 394: 10929 8533 15300 1943 2871 3355 8440 1643 4780 1540210696 4627 2660 3426 1295 1614 466 18055 18870 5343 6172 13204 250914838 16696 14719 12186 9237 5229 14362 18820 11703 10441 9402 3427 69046394 15444 13539 480 4107 3143 11612 16378 9125 5638 5458 4628 1283016118 11849 7565 14432 395: 17920 6614 14177 8433 18119 14743 1547 27984829 13098 1792 7185 5546 12686 17738 14286 17308 15203 7356 15424 889810366 396: 1612 16074 15966 6215 9512 6739 712 4754 9542 926 15250 170943886 1518 14141 3582 17743 2596 16577 7981 8476 11139 9991 7519 165234476 3159 2443 397: 6955 17338 4807 13366 13886 3990 12822 4206 112044966 12012 18854 18590 18505 5937 6734 2714 7052 1150 4851 7568 108705135 3950 2713 870 17666 8862 972 4428 1180 15407 398: 10762 10763 1783118710 4571 2491 18826 7281 7980 12839 7132 3515 11431 13508 15105 1115012370 14765 7924 8625 10099 5689 10100 7454 10434 8719 3324 3759 13967985 1985 399: 6493 10378 18309 8131 4746 2656 3568 15048 5685 902116845 8807 18908 2035 18616 19229 9844 10776 11514 15103 1336 4804 1720414756 12584 18287 12312 5524 13410 6108 12653 16440 10889 4657 450811491 4103 400: 14710 10682 11646 10632 9092 7695 1867 15667 2172 1492010057 4313 15838 6000 16168 13497 12464 7428 3227 15903 11817 6518 723015193 14479 15307 9534 8999 10201 401: 4269 415 5196 1888 17473 594017913 2427 4192 894 2283 3972 4409 4808 15898 14757 16253 13957 402:8349 2740 7671 12717 5139 12414 4134 7424 16056 14823 403: 1589 1760412728 8019 2032 1121 5422 4393 814 3576 1329 18252 17534 7522 6755 855513532 14465 5342 14260 12366 13545 11833 15130 16303 3048 15435 1254812465 9395 4550 15303 15548 16379 15934 3788 11207 10866 16567 979610597 2843 9814 6036 13084 15810 18073 12289 2610 8431 8133 10743 52775274 9812 9270 16062 1410 18832 3278 11379 7651 8619 4074 13579 112787552 3482 1921 3110 13040 3715 588 16215 14079 15855 15077 12165 415814545 7138 12317 14284 16674 7176 4557 4843 4827 14556 15959 12712 1269312300 9895 9617 18976 3521 15031 1253 9850 1713 948 17470 9817 824614610 14409 4404 18149 18916 3122 6477 11641 5344 5193 15313 4566 48566382 6385 8287 13657 6526 12895 13000 17835 5551 4058 4427 6492 392515887 6882 13235 2054 11583 8492 7588 15092 977 3464 6674 11033 1056112912 12978 14142 19136 951 13846 6528 11069 16860 10474 14237 101789206 9999 12994 1371 1369 18499 15946 14691 5796 15067 13591 14544 101169572 9076 3395 18068 9445 17137 10070 12598 12451 5291 8421 13493 1244616898 3422 12469 9327 12751 1725 12393 11867 16709 6201 2489 19025 1535512432 10704 10788 15175 13881 15050 11031 2284 16846 8929 13843 1587712575 2501 3898 8155 8181 13467 7071 13380 18512 6767 11884 3330 1568916649 4862 2400 11639 2206 6774 7277 10750 13838 5371 9923 6934 1197417225 3561 17146 15437 5307 6795 8299 5491 4199 12086 13575 6650 1245212843 7943 4725 8051 12281 12760 4561 2302 8823 7236 18407 18993 139518587 10428 1484 9297 4090 6052 746 5639 5240 11345 8048 13454 3013 37723177 2098 1828 5023 13427 15578 11178 5602 5276 14414 2819 615 9528 39375441 9527 18009 11254 11735 1651 10853 8300 12353 10928 8198 10913 1195411949 16408 11950 8525 8047 17449 14589 10655 14519 9626 17814 7897 30294632 16072 1825 10344 16933 7125 16977 487 5370 1337 10164 7994 148634232 4289 11276 8180 14167 10838 18522 10765 13322 2506 18787 776 165296541 4678 5631 12943 17667 6970 19144 3198 7846 2492 15690 17586 133014379 2818 10177 3242 3316 949 9487 8259 11112 6256 10981 15481 1159014273 5962 3698 13133 1978 10200 2281 15318 15337 15317 1960 10987 1095910458 6605 1442 14285 16347 8163 9798 2999 6258 594 19224 11771 3948 31918283 17226 10012 7302 16116 14915 6717 19232 2868 18598 8016 9887 12616404: 4607 2496 4440 11086 9902 13066 10299 13067 10058 7029 11937 112158917 18762 8922 6629 3169 4418 7255 17978 6392 4006 16582 4177 1329918105 19050 1884 8982 18462 13743 10049 17020 1107 14164 17443 3211 8079415 7769 2665 7716 9819 4793 8194 4487 9462 1406 8450 11140 4167 182033917 15866 2776 1567 9782 15174 12626 4681 12824 14018 4910 4659 25622122 2788 9880 2428 871 8389 7192 16613 2982 3929 11900 8750 5259 1851714460 1370 18379 7537 11555 4777 12893 14616 15017 5702 12071 2926 20182392 18128 17823 17870 13449 12192 1385 8320 11784 8264 14559 5910 117928248 1361 17542 1038 17221 1250 12347 10203 15635 7181 7956 13405 162710660 3003 6686 18312 14977 2919 13737 1505 4646 9493 11913 9654 1441316822 11895 12844 7146 17760 12372 14263 18841 10587 16428 18770 1226416337 14633 11408 17098 2635 7437 18818 16147 17848 15393 9910 6063 57256571 5134 17464 10392 6472 17903 405: 3906 4734 13745 7402 14874 764406: 5355 6984 4667 17305 11490 16367 8426 10381 1464 11818 624 74123333 9382 15475 11760 13337 407: 12506 19148 10381 2347 9031 1569 408:1173 2332 4489

Example 3

This example illustrates the construction of a consensus amino acidsequence of homologous proteins of homologous genes that impart anenhanced trait.

ClustalW program was selected for multiple sequence alignments of theamino acid sequence of SEQ ID NO: 406 and 17 homologs. Three majorfactors affecting the sequence alignments dramatically are (1) proteinweight matrices; (2) gap open penalty; (3) gap extension penalty.Protein weight matrices available for ClustalW program include Blosum,Pam and Gonnet series. Those parameters with gap open penalty and gapextension penalty were extensively tested. On the basis of the testresults, Blosum weight matrix, gap open penalty of 10 and gap extensionpenalty of 1 were chosen for multiple sequence alignment. Shown in FIGS.1A-1D are the sequences of SEQ ID NO: 406, its homologs and theconsensus sequence, as set forth in SEQ ID NO: 19248. The symbols forconsensus sequence are (1) uppercase letters for 100% identity in allpositions of multiple sequence alignment output; (2) lowercase lettersfor >=70% identity; symbol; (3) “X” indicated <70% identity; (4) dashes“-” meaning that gaps were in >=70% sequences.

The consensus amino acid sequence can be used to identify DNAcorresponding to the full scope of this invention that is useful inproviding transgenic plants, for example corn and soybean plants withenhanced agronomic traits, for example improved nitrogen use efficiency,improved yield, improved water use efficiency and/or improved growthunder cold stress, due to the expression in the plants of DNA encoding aprotein with amino acid sequence identical to the consensus amino acidsequence.

Example 4

This example illustrates the identification of amino acid domain by Pfamanalysis.

The amino acid sequence of the expressed proteins that were shown to beassociated with an enhanced trait were analyzed for Pfam protein familyagainst the current Pfam collection of multiple sequence alignments andhidden Markov models using the HMMER software in the appended computerlisting. The Pfam protein families for the proteins of SEQ ID NO: 205through 408 are shown in Table 16. The Hidden Markov model databases forthe identified patent families are also in the appended computer listingallowing identification of other homologous proteins and their cognateencoding DNA to enable the full breadth of the invention for a person ofordinary skill in the art. Certain proteins are identified by a singlePfam domain and others by multiple Pfam domains. For instance, theprotein with amino acids of SEQ ID NO: 214 is characterized by two Pfamdomains, i.e. “C1_4” and “Ssl1”. See also the protein with amino acidsof SEQ ID NO:222 which is characterized by two copies of the Pfam domain“MatE”. In Table 16 “score” is the gathering score for the Hidden MarkovModel of the domain which exceeds the gathering cutoff reported in Table17.

TABLE 16 PEP SEQ ID Pfam domain NO name begin stop score E-value 206zf-A20 10 34 35 2.40E−07  206 zf-AN1 104 144 64.5 3.10E−16  207 Cyclin_N33 168 12.1 0.00023 208 Gpi16 9 603 1272 0 212 BSD 100 167 44.72.80E−10  212 BSD 179 244 67.5 3.90E−17  213 Catalase 18 401 977.73.80E−291 214 Ssl1 22 277 646.3 2.20E−191 214 C1_4 361 409 101.12.90E−27  215 Bromodomain 119 208 105.8 1.10E−28  216 Pkinase 75 343−9.1 1.60E−07  217 PTR2 133 544 197.2 3.50E−56  218 FTCD_N 5 195 377.61.70E−110 219 Abhydrolase_1 50 272 30.4 3.00E−06  220 HhH-GPD 172 31788.2 2.30E−23  222 MatE 40 200 121.9 1.60E−33  222 MatE 261 433 961.00E−25  224 ubiquitin 48 120 24.7 0.00029 224 BAG 138 219 94.23.50E−25  225 Lipase_GDSL 37 365 370.9 1.80E−108 226 DUF231 280 449234.8 1.60E−67  227 Aldedh 16 485 842 2.80E−250 228 RRM_1 110 181 79.97.10E−21  228 RRM_1 214 284 87.8 2.90E−23  229 GATase_2 2 162 10.57.60E−12  229 Asn_synthase 211 478 335.9 6.10E−98  230 zf-C2H2 273 29530.6 5.00E−06  231 HLH 62 113 39 1.50E−08  232 PP2C 39 323 106.95.20E−29  233 zf-C3HC4 96 137 33.4 7.10E−07  234 AP2 83 146 144.52.60E−40  235 Homeobox 84 145 72.6 1.10E−18  236 zf-C3HC4 87 129 31.82.10E−06  237 Acyl_transf_1 52 354 −13.2 6.40E−11  238 Aldo_ket_red 5292 465.6 5.50E−137 239 adh_short 13 179 55.1 2.00E−13  240 HMG_CoA_synt5 453 992.9 1.00E−295 241 SRF-TF 9 59 121.8 1.80E−33  241 K-box 75 175148.8 1.20E−41  242 Ribosomal_S8e 1 237 327.1 2.70E−95  243 zf-C3HC4 103144 37.8 3.30E−08  244 zf-C2H2 4 26 22.3 0.0015 244 zf-C2H2 204 226 22.40.0014 244 zf-C2H2 236 258 20.6 0.005 245 Aa_trans 45 439 358 1.30E−104246 LEA_4 71 141 15.6 0.05 247 p450 66 499 209.4 7.60E−60  248 p450 40503 289.8 4.70E−84  249 Phi_1 25 278 515 7.40E−152 250 zf-B_box 3 4756.2 9.50E−14  251 PP2C 84 348 267.9 1.80E−77  254 Radical_SAM 171 33777.3 4.40E−20  255 Pkinase 25 283 174.5 2.30E−49  256 adh_short 6 18242.9 9.80E−10  257 Skp1_POZ 4 64 105.1 1.80E−28  257 Skp1 112 190 1736.70E−49  258 DPBB_1 73 151 142.6 9.30E−40  258 Pollen_allerg_1 162 239163.9 3.70E−46  259 LRRNT_2 18 58 40.7 4.40E−09  259 LRR_1 86 108 12.31.6 259 LRR_1 109 133 15.7 0.15 259 LRR_1 134 157 17.4 0.047 259 LRR_1158 177 8.3 11 259 LRR_1 179 202 12.5 1.3 259 Pkinase 334 601 3.82.90E−08  260 Methyltransf_11 135 225 25.7 0.00015 261 Ubie_methyltran34 287 368.5 9.10E−108 261 Methyltransf_11 88 205 70.4 5.00E−18  261Methyltransf_12 88 203 38.6 1.90E−08  262 MtN3_slv 11 100 69.4 9.90E−18 262 MtN3_slv 135 221 122.5 1.10E−33  263 Cys_Met_Meta_PP 88 460 768.43.90E−228 263 Beta_elim_lyase 129 376 −107.7 0.0016 264 PAR1 1 181 469.53.60E−138 265 Chal_sti_synt_C 350 493 13.4 0.00012 266 Cyclin_N 43 195113.1 7.30E−31  267 Pkinase 21 418 193.4 5.00E−55  268 Brix 96 271 216.45.60E−62  269 tRNA-synt_2b 81 250 166.9 4.70E−47  269 HGTP_anticodon 402486 17.1 0.00088 270 Proteasome 38 233 81.4 2.50E−21  271Ammonium_transp 19 423 597.5 1.10E−176 272 AUX_IAA 8 204 332.3 7.20E−97 273 Lectin_legB 25 215 −14.8 1.60E−09  273 Lectin_legA 236 279 313.70E−06  273 Pkinase 355 624 179.3 8.50E−51  273 Pkinase_Tyr 355 624116.1 9.20E−32  274 PGK 2 395 675.6 3.30E−200 275 Sugar_tr 27 491 416.72.90E−122 275 MFS_1 31 467 75.7 1.30E−19  276 Hpt 30 112 54.9 2.30E−13 278 RRN3 37 620 1128.7 0 279 Sugar_tr 89 546 595 6.00E−176 279 MFS_1 93505 100.6 4.10E−27  280 AP2 25 88 133.6 4.70E−37  281 Pkinase 4 205 281.20E−09  282 ADH_N 33 119 53.8 5.30E−13  282 ADH_zinc_N 155 318 43.18.50E−10  283 ADH_N 34 149 129.2 1.00E−35  283 ADH_zinc_N 180 315 119.11.10E−32  284 ADH_N 40 165 112.8 8.90E−31  284 ADH_zinc_N 196 338 125.21.60E−34  285 ADH_N 43 173 105.4 1.50E−28  285 ADH_zinc_N 204 348 112.51.10E−30  286 Mov34 23 133 136.1 8.40E−38  287 RRM_1 36 107 101.52.20E−27  288 RRM_1 36 107 73 8.20E−19  289 FA_desaturase 54 269 1351.80E−37  290 Ras 15 176 336.7 3.50E−98  291 SAC3_GANP 24 209 128.91.20E−35  292 RNA_pol_L 6 83 75.5 1.50E−19  293 GSHPx 8 117 234.42.20E−67  294 FKBP_C 115 214 127.7 3.00E−35  295 Ras 10 171 3397.10E−99  296 UQ_con 15 148 81.4 2.50E−21  297 Proteasome 28 215 246.54.90E−71  298 Ras 14 175 317.4 2.20E−92  299 Ldh_1_N 83 226 247.52.40E−71  299 Ldh_1_C 228 394 199.8 5.50E−57  300 Ras 8 209 221.51.60E−63  301 ThiF 30 167 −12.5 3.50E−05  303 Sterol_desat 10 215 128.31.90E−35  304 Sterol_desat 12 227 195.1 1.50E−55  306 Enolase_N 3 133227.6 2.40E−65  306 Enolase_C 138 424 567.5 1.10E−167 307NTP_transferase 4 267 89.5 9.20E−24  308 NAD_binding_2 16 185 −39.11.10E−05  308 NAD_Gly3P_dh_N 17 154 −24.6 0.00015 308 F420_oxidored 18265 341.2 1.60E−99  309 F420_oxidored 4 255 278.2 1.40E−80  310 ADH_N 27145 115.5 1.40E−31  310 ADH_zinc_N 175 318 138.1 2.10E−38  311Aminotran_1_2 44 399 178.8 1.20E−50  312 Aldedh 11 476 903.9 6.50E−269313 NTP_transferase 5 269 52.4 1.40E−13  314 PFK 6 281 515.1 7.10E−152315 NTP_transferase 10 288 20.8 2.90E−11  316 Aminotran_1_2 114 480492.5 4.40E−145 317 NTP_transferase 94 349 346.3 4.40E−101 318 Aldedh 13475 734.7 5.60E−218 319 Aminotran_3 113 448 471.8 7.60E−139 320Cys_Met_Meta_PP 117 395 −276.6 0.0042 320 Aminotran_1_2 118 471 184.52.30E−52  321 Aminotran_1_2 121 483 125.6 1.20E−34  322 DAO 214 489−32.3 0.0011 322 Pyr_redox_2 214 474 69.5 9.70E−18  322 Pyr_redox 343433 55.7 1.40E−13  323 PGM_PMM_I 127 272 140.6 3.80E−39  323 PGM_PMM_II297 407 69.6 8.70E−18  323 PGM_PMM_III 408 528 105.7 1.20E−28  323PGM_PMM_IV 543 632 55.6 1.50E−13  324 Aminotran_3 192 522 634.11.00E−187 325 Biotin_lipoyl 91 164 82.6 1.10E−21  325 E3_binding 198 23474.5 3.00E−19  325 2-oxoacid_dh 262 493 486.5 2.70E−143 326Biotin_lipoyl 91 164 79.5 9.50E−21  326 E3_binding 198 234 76.76.40E−20  326 2-oxoacid_dh 261 492 483.1 2.90E−142 327 Transaldolase 101401 580 2.00E−171 328 Biotin_lipoyl 92 165 88.9 1.40E−23  328 E3_binding201 237 69 1.40E−17  328 2-oxoacid_dh 261 491 478.4 7.90E−141 329cNMP_binding 103 191 73.7 5.30E−19  330 HI0933_like 90 415 −239.1 0.0006330 FAD_binding_2 91 401 −113.1 0.0016 330 GIDA 91 420 −208 0.00018 330DAO 91 353 −32.7 0.0012 330 Pyr_redox_2 91 399 237.8 2.10E−68  330Pyr_redox 261 354 100.9 3.30E−27  330 Pyr_redox_dim 428 537 164.91.90E−46  331 GIDA 94 435 −211.8 0.00032 331 Pyr_redox_2 94 413 217.13.40E−62  331 Pyr_redox 271 368 107.5 3.60E−29  331 Pyr_redox_dim 443552 193.7 3.90E−55  332 FAD_binding_2 99 410 −122.5 0.0042 332Pyr_redox_2 99 409 275 1.30E−79  332 Pyr_redox 266 362 114.7 2.30E−31 332 Pyr_redox_dim 437 546 202.7 7.40E−58  333 Biotin_lipoyl 92 165 81.42.40E−21  333 E3_binding 202 238 68.7 1.60E−17  333 2-oxoacid_dh 261 491485.1 7.30E−143 334 DUF6 117 242 57.9 3.00E−14  334 DUF250 251 397 196.55.70E−56  335 Alpha-amylase 61 489 −62.8 4.70E−06  336 GAF 141 304 86.66.80E−23  336 Phytochrome 315 501 33 8.10E−07  336 HWE_HK 520 600 115.81.10E−31  336 Response_reg 732 848 36.6 7.80E−08  337 NIR_SIR_ferr 69137 83 8.50E−22  337 NIR_SIR 169 332 206.4 5.80E−59  337 NIR_SIR_ferr348 419 75.4 1.50E−19  338 Fer4 9 32 9.4 0.011 338 Fer4 177 202 9.10.012 339 PGI 52 541 1099 0 340 Molybdop_Fe4S4 2 68 80.6 4.30E−21  340Molybdopterin 70 501 390.7 1.90E−114 340 Molydop_binding 627 738 174.91.80E−49  341 PGI 55 545 751.6 4.50E−223 342 Iso_dh 28 469 379.16.10E−111 343 NIR_SIR_ferr 78 147 71.9 1.80E−18  343 NIR_SIR 179 338199.5 6.80E−57  343 NIR_SIR_ferr 354 425 67.3 4.50E−17  344 DUF783 27236 348.6 9.00E−102 346 PA 43 144 89.4 9.50E−24  346 zf-C3HC4 232 27334.7 3.00E−07  347 Peptidase_C26 13 248 158.9 1.10E−44  348 SURF5 17 143252 1.10E−72  349 DUF962 7 171 329.8 4.20E−96  350 Cyclin_N 191 318198.5 1.40E−56  350 Cyclin_C 320 447 173.7 4.00E−49  351 WRKY 237 297137.6 2.90E−38  352 SBDS 6 244 261.6 1.40E−75  353 DUF167 38 112 82.31.40E−21  355 zf-CCCH 2 25 30.5 5.40E−06  356 DIM1 5 139 78.9 1.40E−20 358 Rieske 100 198 68.5 1.90E−17  359 Fe_bilin_red 112 333 416.63.20E−122 360 Radical_SAM 123 282 47.3 4.70E−11  361 LEA_4 107 180 55.31.80E−13  363 Pribosyltran 92 236 146.3 7.30E−41  364 Rhodanese 254 367102 1.60E−27  367 DRMBL 248 368 129 1.20E−35  368 MIP 30 251 276.93.60E−80  369 EBP 23 219 362.2 7.50E−106 371 CcmH 1 139 16.6 6.30E−09 373 CTP_transf_2 30 170 48.4 2.20E−11  374 AP2 46 109 145.7 1.10E−40 375 HLH 48 97 52.3 1.40E−12  376 Myb_DNA-binding 59 104 58.1 2.50E−14 377 Ham1p_like 12 188 153.2 6.10E−43  378 SRF-TF 9 59 112.2 1.40E−30 378 K-box 74 174 76.9 5.80E−20  379 Response_reg 10 152 75.9 1.20E−19 380 Nuc_sug_transp 105 341 17.6 1.50E−14  381 SRF-TF 9 59 105.7 1.20E−28381 K-box 70 167 6.3 0.00011 382 E2F_TDP 16 81 111.2 2.60E−30  382E2F_TDP 151 231 128 2.30E−35  384 RNA_pol_Rpb6 61 114 86.8 5.90E−23  386Myb_DNA-binding 45 96 41.3 2.90E−09  387 zf-C3HC4 83 122 41.3 2.90E−09 388 zf-C3HC4 42 86 35.1 2.20E−07  389 AUX_IAA 11 234 381.5 1.10E−111 390Myb_DNA-binding 14 61 49.1 1.30E−11  390 Myb_DNA-binding 67 112 38.42.10E−08  391 AP2 49 121 137.7 2.80E−38  391 AP2 151 215 101.8 1.80E−27 392 TCP 56 241 161.1 2.60E−45  393 zf-C3HC4 383 424 43.1 8.30E−10  394SET 109 238 182.9 7.10E−52  395 zf-C2H2 36 59 18 0.031 396 zf-C3HC4 206246 34.5 3.40E−07  397 Myb_DNA-binding 26 75 34.3 3.70E−07  397Myb_DNA-binding 134 181 53.7 5.30E−13  398 bZIP_1 171 234 26.9 6.50E−05 398 bZIP_2 171 221 25.3 0.00019 399 zf-C3HC4 111 152 37.5 4.10E−08  400WD40 15 51 16.6 0.079 400 WD40 59 95 23.3 0.00075 400 WD40 210 246 16.40.093 400 WD40 329 366 21.2 0.0033 401 AP2 15 80 89.1 1.20E−23  402 Arm35 75 34.2 4.00E−07  402 Arm 297 337 21.5 0.0027 403 Aminotran_3 42 384494.7 9.80E−146 404 iPGM_N 3 383 716.6 1.60E−212 404 Metalloenzyme 393512 147.2 3.80E−41  405 DUF231 239 408 227.7 2.30E−65  406 DUF231 231404 334.6 1.50E−97  407 DUF231 237 405 336.4 4.40E−98  408 NPH3 204 452364.6 1.40E−106

TABLE 17 Pfam domain accession gathering name number cutoff domaindescription 2-oxoacid_dh PF00198.12 −112 2-oxoacid dehydrogenasesacyltransferase (catalytic domain) ADH_N PF08240.1 −14.5 Alcoholdehydrogenase GroES-like domain ADH_zinc_N PF00107.15 23.8 Zinc-bindingdehydrogenase AP2 PF00847.9 0 AP2 domain AUX_IAA PF02309.6 −83 AUX/IAAfamily Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporterprotein Abhydrolase_1 PF00561.9 5.5 alpha/beta hydrolase foldAcyl_transf_1 PF00698.10 −120 Acyl transferase domain Aldedh PF00171.11−295 Aldehyde dehydrogenase family Aldo_ket_red PF00248.10 −97 Aldo/ketoreductase family Alpha-amylase PF00128.11 −93 Alpha amylase, catalyticdomain Aminotran_1_2 PF00155.9 −57.5 Aminotransferase class I and IIAminotran_3 PF00202.10 −207.6 Aminotransferase class-III Ammonium_transpPF00909.10 −144 Ammonium Transporter Family Arm PF00514.11 40.1Armadillo/beta-catenin-like repeat Asn_synthase PF00733.10 −52.8Asparagine synthase BAG PF02179.5 25 BAG domain BSD PF03909.6 25 BSDdomain Beta_elim_lyase PF01212.10 −114.4 Beta-eliminating lyaseBiotin_lipoyl PF00364.11 −2.3 Biotin-requiring enzyme Brix PF04427.711.4 Brix domain Bromodomain PF00439.13 8.9 Bromodomain C1_4 PF07975.125 TFIIH C1-like domain CTP_transf_2 PF01467.15 −11.8Cytidylyltransferase Catalase PF00199.8 −229 Catalase CcmH PF03918.4−30.8 Cytochrome C biogenesis protein Chal_sti_synt_C PF02797.5 −6.1Chalcone and stilbene synthases, C-terminal domain Cyclin_C PF02984.7−13 Cyclin, C-terminal domain Cyclin_N PF00134.12 −14.7 Cyclin,N-terminal domain Cys_Met_Meta_PP PF01053.9 −278.4 Cys/Met metabolismPLP-dependent enzyme DAO PF01266.11 −36.5 FAD dependent oxidoreductaseDIM1 PF02966.6 25 Mitosis protein DIM1 DPBB_1 PF03330.7 30 Rarelipoprotein A (RIpA)-like double-psi beta- barrel DRMBL PF07522.3 25 DNArepair metallo-beta-lactamase DUF167 PF02594.6 25 Uncharacterized ACR,YggU family COG1872 DUF231 PF03005.5 −58 Arabidopsis proteins of unknownfunction DUF250 PF03151.6 125 Domain of unknown function, DUF250 DUF6PF00892.9 30 Integral membrane protein DUF6 DUF783 PF05615.2 25 Proteinof unknown function (DUF783) DUF962 PF06127.1 25 Protein of unknownfunction (DUF962) E2F_TDP PF02319.9 17 E2F/DP family winged-helixDNA-binding domain E3_binding PF02817.6 10 e3 binding domain EBPPF05241.1 25 Emopamil binding protein Enolase_C PF00113.11 −34 Enolase,C-terminal TIM barrel domain Enolase_N PF03952.5 −4 Enolase, N-terminaldomain F420_oxidored PF03807.5 −34.5 NADP oxidoreductase coenzyme F420-dependent FAD_binding_2 PF00890.13 −124.8 FAD binding domainFA_desaturase PF00487.13 −46 Fatty acid desaturase FKBP_C PF00254.16−7.6 FKBP-type peptidyl-prolyl cis-trans isomerase FTCD_N PF07837.2−67.7 Formiminotransferase domain, N-terminal subdomain Fe_bilin_redPF05996.2 25 Ferredoxin-dependent bilin reductase Fer4 PF00037.14 84Fe-4S binding domain GAF PF01590.14 23 GAF domain GATase_2 PF00310.10−106.2 Glutamine amidotransferases class-II GIDA PF01134.11 −226.7Glucose inhibited division protein A GSHPx PF00255.9 −16 Glutathioneperoxidase Gpi16 PF04113.3 −207.8 Gpi16 subunit, GPI transamidasecomponent HGTP_anticodon PF03129.9 −2 Anticodon binding domainHI0933_like PF03486.4 −255.8 HI0933-like protein HLH PF00010.15 8.2Helix-loop-helix DNA-binding domain HMG_CoA_synt PF01154.7 −230Hydroxymethylglutaryl-coenzyme A synthase HWE_HK PF07536.4 25 HWEhistidine kinase Ham1p_like PF01725.6 −46 Ham1 family HhH-GPD PF00730.1313.5 HhH-GPD superfamily base excision DNA repair protein HomeoboxPF00046.17 −4.1 Homeobox domain Hpt PF01627.11 25 Hpt domain Iso_dhPF00180.9 −97 Isocitrate/isopropylmalate dehydrogenase K-box PF01486.7 0K-box region LEA_4 PF02987.6 25 Late embryogenesis abundant proteinLRRNT_2 PF08263.1 18.6 Leucine rich repeat N-terminal domain LRR_1PF00560.20 19 Leucine Rich Repeat Ldh_1_C PF02866.6 −13 lactate/malatedehydrogenase, alpha/beta C- terminal domain Ldh_1_N PF00056.11 −31.3lactate/malate dehydrogenase, NAD binding domain Lectin_legA PF00138.719 Legume lectins alpha domain Lectin_legB PF00139.9 −77 Legume lectinsbeta domain Lipase_GDSL PF00657.11 10.9 GDSL-like Lipase/AcylhydrolaseMFS_1 PF07690.4 23.5 Major Facilitator Superfamily MIP PF00230.8 −62Major intrinsic protein MatE PF01554.8 59.6 MatE Metalloenzyme PF01676.7−14.4 Metalloenzyme superfamily Methyltransf_11 PF08241.1 17.1Methyltransferase domain Methyltransf_12 PF08242.1 21.4Methyltransferase domain Molybdop_Fe4S4 PF04879.5 13.6 Molybdopterinoxidoreductase Fe4S4 domain Molybdopterin PF00384.11 −50 Molybdopterinoxidoreductase Molydop_binding PF01568.10 1.1 Molydopterin dinucleotidebinding domain Mov34 PF01398.10 −4 Mov34/MPN/PAD-1 family MtN3_slvPF03083.5 −0.8 MtN3/saliva family Myb_DNA-binding PF00249.18 19.1Myb-like DNA-binding domain NAD_Gly3P_dh_N PF01210.12 −44 NAD-dependentglycerol-3-phosphate dehydrogenase N-terminus NAD_binding_2 PF03446.4−63.5 NAD binding domain of 6-phosphogluconate dehydrogenase NIR_SIRPF01077.10 −25 Nitrite and sulphite reductase 4Fe-45 domain NIR_SIR_ferrPF03460.5 20 Nitrite/Sulfite reductase ferredoxin-like half domain NPH3PF03000.4 25 NPH3 family NTP_transferase PF00483.12 −90.5 Nucleotidyltransferase Nuc_sug_transp PF04142.5 −92.4 Nucleotide-sugar transporterPA PF02225.10 13 PA domain PAR1 PF06521.1 25 PAR1 protein PFK PF00365.9−132 Phosphofructokinase PGI PF00342.8 −168.9 Phosphoglucose isomerasePGK PF00162.8 −95.1 Phosphoglycerate kinase PGM_PMM_I PF02878.5 −37.5Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain IPGM_PMM_II PF02879.5 −20 Phosphoglucomutase/phosphomannomutase,alpha/beta/alpha domain II PGM_PMM_III PF02880.5 −11Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain IIIPGM_PMM_IV PF00408.9 −6 Phosphoglucomutase/phosphomannomutase,C-terminal domain PP2C PF00481.10 −44 Protein phosphatase 2C PTR2PF00854.11 −50 POT family Peptidase_C26 PF07722.2 25 Peptidase C26 Phi_1PF04674.2 25 Phosphate-induced protein 1 conserved region PhytochromePF00360.9 11 Phytochrome region Pkinase PF00069.14 −70.8 Protein kinasedomain Pkinase_Tyr PF07714.4 65 Protein tyrosine kinase Pollen_allerg_1PF01357.10 17.2 Pollen allergen Pribosyltran PF00156.14 2 Phosphoribosyltransferase domain Proteasome PF00227.14 −36.7 Proteasome A-type andB-type Pyr_redox PF00070.16 5 Pyridine nucleotide-disulphideoxidoreductase Pyr_redox_2 PF07992.2 −20 Pyridine nucleotide-disulphideoxidoreductase Pyr_redox_dim PF02852.11 −13 Pyridinenucleotide-disulphide oxidoreductase, dimerisation domain RNA_pol_LPF01193.11 16.9 RNA polymerase Rpb3/Rpb11 dimerisation domainRNA_pol_Rpb6 PF01192.12 25 RNA polymerase Rpb6 RRM_1 PF00076.10 15.2 RNArecognition motif. (a.k.a. RRM, RBD, or RNP domain) RRN3 PF05327.1 25RNA polymerase I specific transcription initiation factor RRN3Radical_SAM PF04055.8 8.4 Radical SAM superfamily Ras PF00071.11 18 Rasfamily Response_reg PF00072.11 −14.4 Response regulator receiver domainRhodanese PF00581.9 25 Rhodanese-like domain Ribosomal_S8e PF01201.11 25Ribosomal protein S8e Rieske PF00355.15 −7 Rieske [2Fe-2S] domainSAC3_GANP PF03399.5 −15.2 SAC3/GANP/Nin1/mts3/elF-3 p25 family SBDSPF01172.7 −78 Shwachman-Bodian-Diamond syndrome (SBDS) proteins SETPF00856.16 15.8 SET domain SRF-TF PF00319.8 11 SRF-type transcriptionfactor (DNA-binding and dimerisation domain) SURF5 PF06179.2 25 Surfeitlocus protein 5 Skp1 PF01466.8 −2 Skp1 family, dimerisation domainSkp1_POZ PF03931.4 14.9 Skp1 family, tetramerisation domain Ssl1PF04056.4 −151.8 Ssl1-like Sterol_desat PF01598.7 −13 Sterol desaturaseSugar_tr PF00083.12 −85 Sugar (and other) transporter TCP PF03634.3 −38TCP family transcription factor ThiF PF00899.10 −38.4 ThiF familyTransaldolase PF00923.8 −49 Transaldolase UQ_con PF00179.15 −30Ubiquitin-conjugating enzyme Ubie_methyltran PF01209.8 −117 ubiE/COQ5methyltransferase family WD40 PF00400.19 21.4 WD domain, G-beta repeatWRKY PF03106.5 25 WRKY DNA-binding domain adh_short PF00106.13 −46.6short chain dehydrogenase bZIP_1 PF00170.10 16.5 bZIP transcriptionfactor bZIP_2 PF07716.4 15 Basic region leucine zipper cNMP_bindingPF00027.17 20.6 Cyclic nucleotide-binding domain iPGM_N PF06415.3 −263.4BPG-independent PGAM N-terminus (iPGM_N) p450 PF00067.11 −105 CytochromeP450 tRNA-synt_2b PF00587.14 −40.5 tRNA synthetase class II core domain(G, H, P, S and T) Ubiquitin PF00240.12 19.4 Ubiquitin family zf-A20PF01754.6 25 A20-like zinc finger zf-AN1 PF01428.6 15 AN1-like Zincfinger zf-B_box PF00643.13 11.1 B-box zinc finger zf-C2H2 PF00096.14 19Zinc finger, C2H2 type zf-C3HC4 PF00097.12 16.9 Zinc finger, C3HC4 type(RING finger) zf-CCCH PF00642.14 10.7 Zinc finger C-x8-C-x5-C-x3-H type(and similar)

Example 5

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic crop plants of this invention. Primers for PCR amplificationof protein coding nucleotides of recombinant DNA are designed at or nearthe start and stop codons of the coding sequence, in order to eliminatemost of the 5′ and 3′ untranslated regions. DNA of interest, i.e. eachDNA identified in Table 1 and the DNA for the identified homologousgenes, are cloned and amplified by PCR prior to insertion into theinsertion site the base vector.

Elements of an exemplary common expression vector, pMON82060 areillustrated in Table 18. The exemplary base vector which is especiallyuseful for corn transformation is illustrated in FIG. 2 and assembledusing technology known in the art. The DNA of interest are inserted in aexpression vector at the insertion site between the intron1 of rice act1 gene and the termination sequence of PinII gene.

TABLE 18 pMON82060 Coordinates of SEQ ID function name annotation NO:19249 Agro B-AGRtu.right border Agro right border sequence, essentialfor 5235-5591 transformation transfer of T-DNA. Gene of P-Os.Act1Promoter from the rice actin gene act1. 5609-7009 interest plantL-Os.Act1 Leader (first exon) from the rice actin 1 expression gene.cassette I-Os.Act1 First intron and flanking UTR exon sequences from therice actin 1 gene insertion site T-St.Pis4 The 3′ non-translated regionof the 7084-8026 potato proteinase inhibitor II gene which functions todirect polyadenylation of the mRNA Plant P-CaMV.35S CaMV 35S promoter8075-8398 selectable L-CaMV.35S 5′ UTR from the 35S RNA of CaMV markerCR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226expression resistance to neomycin and kanamycin cassette T-AGRtu.nos A3′ non-translated region of the 9255-9507 nopaline synthase gene ofAgrobacterium tumefaciens Ti plasmid which functions to directpolyadenylation of the mRNA. . Agro B-AGRtu.left border Agro left bordersequence, essential for  39-480 transformation transfer of T-DNA.Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication from567-963 in E. coli plasmid RK.2. CR-Ec.rop Coding region for repressorof primer 2472-2663 from the ColE1 plasmid. Expression of this geneproduct interferes with primer binding at the origin of replication,keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin ofreplication from 3091-3679 the E. coli plasmid ColE1. P-Ec.aadA-SPC/STRpromoter for Tn7 adenylyltransferase 4210-4251 (AAD(3″)) CR-Ec.aadA-Coding region for Tn7 4252-5040 SPC/STR adenylyltransferase (AAD(3″))conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR3′ UTR from the Tn7 adenylyltransferase 5041-5098 (AAD(3″)) gene of E.coli.

Plasmids for use in transformation of soybean are also prepared.Elements of an exemplary common expression vector plasmid pMON82053 areshown in Table 19 below. This exemplary soybean transformation basevector illustrated in FIG. 3 was assembled using the technology known inthe art. DNA of interest, i.e. each DNA identified in Table 1 and theDNA for the identified homologous genes, are cloned and amplified by PCRprior to insertion into the insertion site the base vector at theinsertion site between the enhanced 35S CaMV promoter and thetermination sequence of cotton E6 gene.

TABLE 19 pMON82053 Coordinates of SEQ ID function name annotation NO:19250 Agro B-AGRtu.left border Agro left border 6144-6585 transforamtionsequence, essential for transfer of T-DNA. Plant P-At.Act7 Promoter fromthe 6624-7861 selectable Arabidopsis actin 7 gene marker L-At.Act7 5′UTRof Arabidopsis expression Act7 gene cassette I-At.Act7 Intron from theArabidopsis actin 7 gene TS-At.ShkG-CTP2 Transit peptide region of7864-8091 Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding8092-9459 CP4.nno_At region with dicot preferred codon usage.T-AGRtu.nos A 3′ non-translated region 9466-9718 of the nopalinesynthase gene of Agrobacterium tumefaciens Ti plasmid which functions todirect polyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for35S RNA  1-613 interest from CaMV containing a expression duplication ofthe −90 to cassette −350 region. insertion site T-Gb.E6-3b 3′untranslated region  688-1002 from the fiber protein E6 gene ofsea-island cotton; Agro B-AGRtu.right border Agro right border 1033-1389transformation sequence, essential for transfer of T-DNA. OR-Ec.oriV-RK2The vegetative origin of 5661-6057 replication from plasmid RK2.CR-Ec.rop Coding region for 3961-4152 repressor of primer from the ColE1plasmid. Expression of this gene product interferes with primer bindingat the origin of replication, keeping plasmid copy number low.Maintenance OR-Ec.ori-ColE1 The minimal origin of 2945-3533 in E. colireplication from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR romoterfor Tn7 2373-2414 adenylyltransferase (AAD(3″)) CR-Ec.aadA-SPC/STRCoding region for Tn7 1584-2372 adenylyltransferase (AAD(3″)) conferringspectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR fromthe Tn7 1526-1583 adenylyltransferase (AAD(3″)) gene of E. coli.

Example 5

This example illustrates monocot plant transformation useful inproducing the transgenic plant cells of this invention by transformationof corn. Corn plants of a readily transformable line are grown in thegreenhouse and ears harvested when the embryos are 1.5 to 2.0 mm inlength. Ears are surface sterilized by spraying or soaking the ears in80% ethanol, followed by air drying. Immature embryos are isolated fromindividual kernels on surface sterilized ears. Prior to inoculation ofmaize cells, Agrobacterium cells are grown overnight at roomtemperature. Immature maize embryos are inoculated with Agrobacteriumshortly after excision, and incubated at room temperature withAgrobacterium for 5-20 minutes. Immature embryos are then co-culturedwith Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-culturedembryos are transferred to selection media and cultured forapproximately two weeks to allow embryogenic callus to develop.Embryogenic callus is transferred to culture medium containing 100 mg/Lparomomycin and subcultured at about two week intervals. Transformantsare recovered 6 to 8 weeks after initiation of selection.

Plasmid vectors are prepared essentially as described in Example 5 fortransforming into corn each of the DNA of interest, i.e. each DNAidentified in Table 1 and the DNA for the identified homologous genes,by Agrobacterium-mediated transformation.

For Agrobacterium-mediated transformation of maize callus, immatureembryos are cultured for approximately 8-21 days after excision to allowcallus to develop. Callus is then incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. The callus and Agrobacterium are co-culturedwithout selection for 3-6 days followed by selection on paromomycin forapproximately 6 weeks, with biweekly transfers to fresh media, andparomomycin resistant callus identified as containing the recombinantDNA in an expression cassette.

To regenerate transgenic corn plants trangenic callus resulting fromtransformation is placed on media to initiate shoot development inplantlets which are transferred to potting soil for initial growth in agrowth chamber at 26 degrees C. followed by a mist bench beforetransplanting to 5 inch pots where plants are grown to maturity. Theplants are self fertilized and seed is harvested for screening as seed,seedlings or progeny R2 plants or hybrids, e.g. for yield trials in thescreens indicated above. Populations of transgenic plants and seedsproduced form transgenic plant cells from each transgenic event arescreened as described in Example 7 below to identify the members of thepopulation having the enhanced trait.

Example 6

This example illustrates dicot plant transformation useful in producingthe transgenic plant cells of this invention by transformation ofsoybean plants. For Agrobacterium mediated transformation, soybean seedsare germinated overnight and the meristem explants excised. Themeristems and the explants are placed in a wounding vessel. Soybeanexplants and induced Agrobacterium cells from a strain containingplasmid DNA with the gene of interest cassette and a plant selectablemarker cassette are mixed no later than 14 hours from the time ofinitiation of seed germination and wounded using sonication. Followingwounding, explants are placed in co-culture for 2-5 days at which pointthey are transferred to selection media for 6-8 weeks to allow selectionand growth of transgenic shoots. Trait positive shoots are harvestedapproximately 6-8 weeks post bombardment and placed into selectiverooting media for 2-3 weeks. Shoots producing roots are transferred tothe greenhouse and potted in soil. Shoots that remain healthy onselection, but do not produce roots are transferred to non-selectiverooting media for an additional two weeks. Roots from any shoots thatproduce roots off selection are tested for expression of the plantselectable marker before they are transferred to the greenhouse andpotted in soil. Populations of transgenic plants and seeds produced formtransgenic plant cells from each transgenic event are screened asdescribed in Example 7 below to identify the members of the populationhaving the enhanced trait.

Example 7

This example illustrates identification of plant cells of the inventionby screening derived plants and seeds for enhanced trait. Manytransgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Populations of transgenic seed and plants prepared in Examples 5and 6 are screened to identify those transgenic events providingtransgenic plant cells with recombinant DNA imparting an enhanced trait.Each population is screened for nitrogen use efficiency, increasedyield, enhanced water use efficiency, enhanced tolerance to cold andheat, enhanced level of oil and protein in seed using assays describedbelow. Plant cells having recombinant DNA with each of the genesidentified in Table 1 and the identified homologs are identified inplants and seeds with at least one of the enhanced traits.

Selection for Enhanced Nitrogen Use Efficiency

The physiological efficacy of transgenic corn plants (tested as hybrids)can be tested for nitrogen use efficiency (NUE) traits in ahigh-throughput nitrogen (N) selection method. The collected data arecompared to the measurements from wildtype controls using a statisticalmodel to determine if the changes are due to the transgene. Raw datawere analyzed by SAS software. Results shown herein are the comparisonof transgenic plants relative to the wildtype controls.

(1) Media Preparation for Planting a NUE Protocol

Planting materials used: Metro Mix 200 (vendor: Hummert) Cat. #10-0325,Scotts Micro Max Nutrients (vendor: Hummert) Cat. #07-6330, OS 4⅓″×3⅞″pots (vendor: Hummert) Cat. #16-1415, OS trays (vendor: Hummert) Cat.#16-1515, Hoagland's macronutrients solution, Plastic 5″ stakes (vendor:Hummert) yellow Cat. #49-1569, white Cat. #49-1505, Labels with numbersindicating material contained in pots. Fill 500 pots to rim with MetroMix 200 to a weight of ˜140 g/pot. Pots are filled uniformly by using abalancer. Add 0.4 g of Micro Max nutrients to each pot. Stir ingredientswith spatula to a depth of 3 inches while preventing material loss.

(2) Planting a NUE Selection in the Greenhouse

(a) Seed Germination—Each pot is lightly atered twice using reverseosmosis purified water. The first watering is scheduled to occur justbefore planting; and the second watering, after the seed has beenplanted in the pot. Ten Seeds of each entry (1 seed per pot) are plantedto select eight healthy uniform seedlings. Additional wild type controlsare planted for use as border rows. Alternatively, 15 seeds of eachentry (1 seed per pot) are planted to select 12 healthy uniformseedlings (this larger number of plantings is used for the second, orconfirmation, planting). Place pots on each of the 12 shelves in theConviron growth chamber for seven days. This is done to allow moreuniform germination and early seedling growth. The following growthchamber settings are 25° C./day and 22° C./night, 14 hours light and tenhours dark, humidity ˜80%, and light intensity ˜350 μmol/m²/s (at potlevel). Watering is done via capillary matting similar to greenhousebenches with duration of ten minutes three times a day.

(b) Seedling transfer—After seven days, the best eight or 12 seedlingsfor the first or confirmation pass runs, respectively, are chosen andtransferred to greenhouse benches. The pots are spaced eight inchesapart (center to center) and are positioned on the benches using thespacing patterns printed on the capillary matting. The Vattex mattingcreates a 384-position grid, randomizing all range, row combinations.Additional pots of controls are placed along the outside of theexperimental block to reduce border effects.

Plants are allowed to grow for 28 days under the low N run or for 23days under the high N run. The macronutrients are dispensed in the formof a macronutrient solution (see composition below) containing preciseamounts of N added (2 mM NH₄NO₃ for limiting N selection and 20 mMNH₄NO₃ for high N selection runs). Each pot is manually dispensed 100 mlof nutrient solution three times a week on alternate days starting ateight and ten days after planting for high N and low N runs,respectively. On the day of nutrient application, two 20 min wateringsat 05:00 and 13:00 are skipped. The vattex matting should be changedevery third run to avoid N accumulation and buildup of root matter.Table 7 shows the amount of nutrients in the nutrient solution foreither the low or high nitrogen selection.

TABLE 22 2 mM NH₄NO₃ 20 mM NH₄NO₃ (high (Low Nitrogen Growth NitrogenGrowth Condition, Low N) Condition, High N) Nutrient Stock mL/L mL/L 1MNH₄N0₃ 2 20 1M KH₂PO₄ 0.5 0.5 1M MgSO₄•7H₂O 2 2 1M CaCl₂ 2.5 2.5 1MK₂SO₄ 1 1 Note: Adjust pH to 5.6 with HCl or KOH

(c) Harvest Measurements and Data Collection—After 28 days of plantgrowth for low N runs and 23 days of plant growth for high N runs, thefollowing measurements are taken (phenocodes in parentheses): totalshoot fresh mass (g) (SFM) measured by Sartorius electronic balance, V6leaf chlorophyll measured by Minolta SPAD meter (relative units) (LC),V6 leaf area (cm²) (LA) measured by a Li-Cor leaf area meter, V6 leaffresh mass (g) (LFM) measured by Sartorius electronic balance, and V6leaf dry mass (g) (LDM) measured by Sartorius electronic balance. Rawdata were analyzed by SAS software. Results shown are the comparison oftransgenic plants relative to the wildtype controls.

To take a leaf reading, samples were excised from the V6 leaf. Sincechlorophyll meter readings of corn leaves are affected by the part ofthe leaf and the position of the leaf on the plant that is sampled, SPADmeter readings were done on leaf six of the plants. Three measurementsper leaf were taken, of which the first reading was taken from a pointone-half the distance between the leaf tip and the collar and halfwayfrom the leaf margin to the midrib while two were taken toward the leaftip. The measurements were restricted in the area from ½ to ¾ of thetotal length of the leaf (from the base) with approximately equalspacing between them. The average of the three measurements was takenfrom the SPAD machine.

Leaf fresh mass is recorded for an excised V6 leaf, the leaf is placedinto a paper bag. The paper bags containing the leaves are then placedinto a forced air oven at 80° C. for 3 days. After 3 days, the paperbags are removed from the oven and the leaf dry mass measurements aretaken.

From the collected data, two derived measurements are made: (1) Leafchlorophyll area (LCA), which is a product of V6 relative chlorophyllcontent and its leaf area (relative units). Leaf chlorophyll area=leafchlorophyll X leaf area. This parameter gives an indication of thespread of chlorophyll over the entire leaf area; (2) specific leaf area(LSA) is calculated as the ratio of V6 leaf area to its dry mass (cm²/gdry mass), a parameter also recognized as a measure of NUE.

Nitrogen Use Field Efficacy Assay

Level I. Transgenic plants provided by the present invention are plantedin field without any nitrogen source being applied. Transgenic plantsand control plants are grouped by genotype and construct with controlsarranged randomly within genotype blocks. Each type of transgenic plantsare tested by 3 replications and across 5 locations. Nitrogen levels inthe fields are analyzed in early April pre-planting by collecting 30sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples areanalyzed for nitrate-nitrogen, phosphorus (P), Potassium (K), organicmatter and pH to provide baseline values. P, K and micronutrients areapplied based upon soil test recommendations.Level II. Transgenic plants provided by the present invention areplanted in field with three levels of nitrogen (N) fertilizer beingapplied, i.e. low level (0 N), medium level (80 lb/ac) and high level(180 lb/ac). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used asthe N source and apply by broadcast boom and incorporate with a fieldcultivator with rear rolling basket in the same direction as intendedcrop rows. Although there is no N applied to the 0 N treatment the soilshould still be disturbed in the same fashion as the treated area.Transgenic plants and control plants are grouped by genotype andconstruct with controls arranged randomly within genotype blocks. Eachtype of transgenic plants is tested by 3 replications and across 4locations. Nitrogen levels in the fields are analyzed in early Aprilpre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus(P), Potassium (K), organic matter and pH to provide baseline values. P,K and micronutrients are applied based upon soil test recommendations.

Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield ascompared to a control plant. Improved yield can result from enhancedseed sink potential, i.e. the number and size of endosperm cells orkernels and/or enhanced sink strength, i.e. the rate of starchbiosynthesis. Sink potential can be established very early during kerneldevelopment, as endosperm cell number and size are determined within thefirst few days after pollination.

Much of the increase in corn yield of the past several decades hasresulted from an increase in planting density. During that period, cornyield has been increasing at a rate of 2.1 bushels/acre/year, but theplanting density has increased at a rate of 250 plants/acre/year. Acharacteristic of modern hybrid corn is the ability of these varietiesto be planted at high density. Many studies have shown that a higherthan current planting density should result in more biomass production,but current germplasm does not perform. well at these higher densities.One approach to increasing yield is to increase harvest index (HI), theproportion of biomass that is allocated to the kernel compared to totalbiomass, in high density plantings.

Effective yield selection of enhanced yielding transgenic corn eventsuses hybrid progeny of the transgenic event over multiple locations withplants grown under optimal production management practices, and maximumpest control. A useful target for improved yield is a 5% to 10% increasein yield as compared to yield produced by plants grown from seed for acontrol plant. Selection methods may be applied in multiple and diversegeographic locations, for example up to 16 or more locations, over oneor more planting seasons, for example at least two planting seasons tostatistically distinguish yield improvement from natural environmentaleffects. It is to plant multiple transgenic plants, positive andnegative control plants, and pollinator plants in standard plots, forexample 2 row plots, 20 feet long by 5 feet wide with 30 inches distancebetween rows and a 3 foot alley between ranges. Transgenic events can begrouped by recombinant DNA constructs with groups randomly placed in thefield. A pollinator plot of a high quality corn line is planted forevery two plots to allow open pollination when using male steriletransgenic events. A useful planting density is about 30,000plants/acre. High planting density is greater than 30,000 plants/acre,preferably about 40,000 plants/acre, more preferably about 42,000plants/acre, most preferably about 45,000 plants/acre. Transgenic cornplants and soybean plants with each recombinant DNA construct preparedin Examples 5 and 6 are identified as exhibiting at least 5% yieldincrease as compared to control plants.

Selection for Enhanced Water Use Efficiency (WUE)

Described in this example is a high-throughput method for greenhouseselection of transgenic corn plants to wild type corn plants (tested asinbreds or hybrids) for water use efficiency. This selection processimposes 3 drought/re-water cycles on plants over a total period of 15days after an initial stress free growth period of 11 days. Each cycleconsists of 5 days, with no water being applied for the first four daysand a water quenching on the 5th day of the cycle. The primaryphenotypes analyzed by the selection method are the changes in plantgrowth rate as determined by height and biomass during a vegetativedrought treatment. The hydration status of the shoot tissues followingthe drought is also measured. The plant height are measured at threetime points. The first is taken just prior to the onset drought when theplant is 11 days old, which is the shoot initial height (SIH). The plantheight is also measured halfway throughout the drought/re-water regimen,on day 18 after planting, to give rise to the shoot mid-drought height(SMH). Upon the completion of the final drought cycle on day 26 afterplanting, the shoot portion of the plant is harvested and measured for afinal height, which is the shoot wilt height (SWH) and also measured forshoot wilted biomass (SWM). The shoot is placed in water at 40 degreeCelsius in the dark. Three days later, the shoot is weighted to giverise to the shoot turgid weight (STM). After drying in an oven for fourdays, the shoots are weighted for shoot dry biomass (SDM). The shootaverage height (SAH) is the mean plant height across the 3 heightmeasurements. The procedure described above may be adjusted for +/−˜oneday for each step given the situation.

To correct for slight differences between plants, a size correctedgrowth value is derived from SIH and SWH. This is the Relative GrowthRate (RGR). Relative Growth Rate (RGR) is calculated for each shootusing the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)*100]. Relative watercontent (RWC) is a measurement of how much (%) of the plant was water atharvest. Water Content (RWC) is calculated for each shoot using theformula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants ofthis age run around 98% RWC.

Selection for Growth Under Cold Stress

(1) Cold germination assay—Three sets of seeds are used for the assay.The first set consists of positive transgenic events (F1 hybrid) wherethe genes of the present invention are expressed in the seed. The secondseed set is nontransgenic, wild-type negative control made from the samegenotype as the transgenic events. The third set consisted of two coldtolerant and one cold sensitive commercial check lines of corn. Allseeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide,Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan isapplied per 45 g of corn seeds by mixing it well and drying thefungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within anindividual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seedsfrom an event are placed into one cell of the germination tray. Eachtray can hold 21 transgenic events and 3 replicates of wildtype(LH244SDms+LH59), which is randomized in a complete block design. Forevery event there are five replications (five trays). The trays areplaced at 9.7 C for 24 days (no light) in a Convrion growth chamber(Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Twohundred and fifty millilters of deionized water are added to eachgermination tray. Germination counts are taken 10th, 11th, 12th, 13th,14th, 17th, 19th, 21st, and 24th day after start date of the experiment.Seeds are considered germinated if the emerged radicle size is 1 cm.From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1−n _(i) ]*[P _(i) −P _(i-1)]))/T

Where T is the total number of days for which the germination assay isperformed. The number of days after planting is defined by n. “i”indicated the number of times the germination had been counted,including the current day. P is the percentage of seeds germinatedduring any given rating. Statistical differences are calculated betweentransgenic events and wild type control. After statistical analysis, theevents that show a statistical significance at the p level of less than0.1 relative to wild-type controls will advance to a secondary coldselection. The secondary cold screen is conducted in the same manner ofthe primary selection only increasing the number of repetitions to ten.Statistical analysis of the data from the secondary selection isconducted to identify the events that show a statistical significance atthe p level of less than 0.05 relative to wild-type controls.

(2) Cold Shock assay—The experimental set-up for the cold shock assay isthe same as described in the above cold germination assay except seedswere grown in potted media for the cold shock assay.

The desired numbers of 2.5″ square plastic pots are placed on flats(n=32, 4×8). Pots were filled with Metro Mix 200 soil-less mediacontaining 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots andFlat are obtained from Hummert International, Earth City, Mo.). Afterplanting seeds, pots are placed in a growth chamber set at 23° C.,relative humidity of 65% with 12 hour day and night photoperiod (300uE/m2-min). Planted seeds are watered for 20 minute every other day bysub-irrigation and flats were rotated every third day in a growthchamber for growing corn seedlings.

On the 10^(th) day after planting the transgenic positive and wild-typenegative (WT) plants are positioned in flats in an alternating pattern.Chlorophyll fluorescence of plants is measured on the 10^(th) day duringthe dark period of growth by using a PAM-2000 portable fluorometer asper the manufacturer's instructions (Walz, Germany). After chlorophyllmeasurements, leaf samples from each event are collected for confirmingthe expression of genes of the present invention. For expressionanalysis six VI leaf tips from each selection are randomly harvested.The flats are moved to a growth chamber set at 5° C. All otherconditions such as humidity, day/night cycle and light intensity areheld constant in the growth chamber. The flats are sub-irrigated everyday after transfer to the cold temperature. On the 4^(th) daychlorophyll fluorescence is measured. Plants are transferred to normalgrowth conditions after six days of cold shock treatment and allowed torecover for the next three days. During this recovery period the lengthof the V3 leaf is measured on the 1^(st) and 3^(rd) days. After two daysof recovery V2 leaf damage is determined visually by estimating percentof green V2 leaf.

Statistical differences in V3 leaf growth, V2 leaf necrosis andfluorescence during pre-shock and cold shock can be used for estimationof cold shock damage on corn plants.

(3) Early seedling growth assay—Three sets of seeds are used for theexperiment. The first set consists of positive transgenic events (F1hybrid) where the genes of the present invention are expressed in theseed. The second seed set is nontransgenic, wild-type negative controlmade from the same genotype as the transgenic events. The third seed setconsists of two cold tolerant and two cold sensitive commercial checklines of corn. All seeds are treated with a fungicide “Captan”,(3a,4,7,a-tetrahydro-2-[(trichloromethly)thio]-1H-isoindole-1,3(2H)-dione,Drex Chemical Co. Memphis, Tenn.). Captan (0.43 mL) was applied per 45 gof corn seeds by mixing it well and drying the fungicide prior to theexperiment.

Seeds are grown in germination paper for the early seedling growthassay. Three 12″×18″ pieces of germination paper (Anchor Paper #SD7606)are used for each entry in the test (three repetitions per transgenicevent). The papers are wetted in a solution of 0.5% KNO₃ and 0.1%Thyram.

For each paper fifteen seeds are placed on the line evenly spaced downthe length of the paper. The fifteen seeds are positioned on the papersuch that the radical would grow downward, for example longer distanceto the paper's edge. The wet paper is rolled up starting from one of theshort ends. The paper is rolled evenly and tight enough to hold theseeds in place. The roll is secured into place with two large paperclips, one at the top and one at the bottom. The rolls are incubated ina growth chamber at 23° C. for three days in a randomized complete blockdesign within an appropriate container. The chamber is set for 65%humidity with no light cycle. For the cold stress treatment the rollsare then incubated in a growth chamber at 12° C. for twelve days. Thechamber is set for 65% humidity with no light cycle.

After the cold treatment the germination papers are unrolled and theseeds that did not germinate are discarded. The lengths of the radicleand coleoptile for each seed are measured through an automated imagingprogram that automatically collects and processes the images. Theimaging program automatically measures the shoot length, root length,and whole seedling length of every individual seedling and thencalculates the average of each roll.

After statistical analysis, the events that show a statisticalsignificance at the p level of less than 0.1 relative to wild-typecontrols will advance to a secondary cold selection. The secondary coldselection is conducted in the same manner of the primary selection onlyincreasing the number of repetitions to five. Statistical analysis ofthe data from the secondary selection is conducted to identify theevents that show a statistical significance at the p level of less than0.05 relative to wild-type controls.

4. Cold Field Efficacy Trial

This example sets forth a cold field efficacy trial to identify geneconstructs that confer enhanced cold vigor at germination and earlyseedling growth under early spring planting field conditions inconventional-till and simulated no-till environments. Seeds are plantedinto the ground around two weeks before local farmers are beginning toplant corn so that a significant cold stress is exerted onto the crop,named as cold treatment. Seeds also are planted under local optimalplanting conditions such that the crop has little or no exposure to coldcondition, named as normal treatment. The cold field efficacy trials arecarried out in five locations, including Glyndon Minn., Mason Mich.,Monmouth Ill., Dayton Iowa, Mystic Conn. At each location, seeds areplanted under both cold and normal conditions with 3 repetitions pertreatment, 20 kernels per row and single row per plot. Seeds are planted1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperaturemonitors are set up at each location to monitor both air and soiltemperature daily.

Seed emergence is defined as the point when the growing shoot breaks thesoil surface. The number of emerged seedling in each plot is countedeveryday from the day the earliest plot begins to emerge until nosignificant changes in emergence occur. In addition, for each plantingdate, the latest date when emergence is 0 in all plots is also recorded.Seedling vigor is also rated at V3-V4 stage before the average of cornplant height reaches 10 inches, with 1=excellent early growth, 5=Averagegrowth and 9=poor growth. Days to 50% emergence, maximum percentemergence and seedling vigor are calculated using SAS software for thedata within each location or across all locations.

Screens for Transgenic Plant Seeds with Increased Protein and/or OilLevels

This example sets forth a high-throughput selection for identifyingplant seeds with improvement in seed composition using the Infratec 1200series Grain Analyzer, which is a near-infrared transmittancespectrometer used to determine the composition of a bulk seed sample.Near infrared analysis is a non-destructive, high-throughput method thatcan analyze multiple traits in a single sample scan. An NIR calibrationfor the analytes of interest is used to predict the values of an unknownsample. The NIR spectrum is obtained for the sample and compared to thecalibration using a complex chemometric software package that provides apredicted values as well as information on how well the sample fits inthe calibration.

Infratec Model 1221, 1225, or 1227 with transport module by Foss NorthAmerica is used with cuvette, item #1000-4033, Foss North America or forsmall samples with small cell cuvette, Foss standard cuvette modified byLeon Girard Co. Corn and soy check samples of varying compositionmaintained in check cell cuvettes are supplied by Leon Girard Co. NITcollection software is provided by Maximum Consulting Inc. Software.Calculations are performed automatically by the software. Seed samplesare received in packets or containers with barcode labels from thecustomer. The seed is poured into the cuvettes and analyzed as received.

TABLE 26 Typical sample(s): Whole grain corn and soybean seedsAnalytical time to run method: Less than 0.75 min per sample Totalelapsed time per run: 1.5 minute per sample Typical and minimum sampleCorn typical: 50 cc; minimum 30 cc size: Soybean typical: 50 cc; minimum5 cc Typical analytical range: Determined in part by the specificcalibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, andprotein 35-50%.

Example 8

This example describes recombinant DNA constructs of the invention,useful for suppressing the expression of a protein identified by Pfam,Catalase, Bromdomain, FTCD_N, MatE, DPBB_1, tRNA-synt_2b, Sugar_tr andMFS_1, DUF6 and DUF250, LEA_4, MIP or DUF231, in a corn or soybeanplant, by expressing a sense and an anti-sense fragment of the nativeDNA encoding the protein, essentially as described in U.S. patentapplication Ser. No. 11/303,745, incorporated herein by reference.Specific gene suppression constructs are targeted to the native gene incorn and soybean plants that are homologs of the genes encoding theprotein with an amino acid sequence of SEQ ID NO:213, 215, 218, 222,258, 269, 275, 334, 361, 368, and 407.

The constructs include a promoter operably linked to DNA thattranscribes to RNA that forms a double stranded RNA in transgenic plantcells for suppressing expression of the protein to provided the enhancedtrait in the corn and soybean plants. Populations of transgenic plantsand seeds derived from the plant cells are screened to identify thoseplants exhibiting the enhanced traits associated with suppression ofthose genes.

1. (canceled)
 2. A plant cell with stably integrated, recombinant DNAcomprising a promoter that is functional in plant cells and that isoperably linked to DNA from a plant, bacteria or yeast that encodes aprotein having at least one domain of amino acids in a sequence thatexceeds the Pfam gathering cutoff for amino acid sequence alignment witha protein domain family identified by a Pfam name in the group of Pfamnames consisting of 2-oxoacid_dh, ADH_N ADH_zinc_N, AP2, AUX_IAA,Aa_trans, Abhydrolase_1, Acyl_transf_1, Aldedh, Aldo_ket_red,Alpha-amylase, Aminotran_1_2, Aminotran_3, Ammonium_tramp, Arm,Asn_synthase, BAG, BSD, Beta_elim_lyase, Biotin_lipoyl, Brix,Bromodomain, C1_4, CTP_transf_2, Catalase, CcmH, Chal_sti_synt_C,Cyclin_C, Cyclin_N, Cys_Met_Meta_PP, DAO, DIM1, DPBB_1, DRMBL, DUF167,DUF231, DUF250, DUF6, DUF783, DUF962, E2F_TDP, E3_binding, EBP,Enolase_C, Enolase_N, F420_oxidored, FAD_binding_2, FA_desaturase,FKBP_C, FTCD_N, Fe_bilin_red, Fer4, GAF, GATase_2, GIDA, GSHPx, Gpi16,HGTP_anticodon, HI0933_like, HLH, HMG_CoA_synt, HWE_HK, Ham1p_like,HhH-GPD, Homeobox, Hpt, Iso_dh, K-box, LEA_4, LRRNT_2, LRR_1, Ldh_1_C,Ldh_1_N, Lectin_legA, Lectin_legB, Lipase_GDSL, MFS_1, MIP, MatE,Metalloenzyme, Methyltransf_11, Methyltransf_12, Molybdop_Fe4S4,Molybdopterin, Molydop_binding, Mov34, MtN3_slv, Myb_DNA-binding,NAD_Gly3P_dh_N, NAD_binding_2, NIR_SIR, NIR_SIR_ferr, NPH3,NTP_transferase, Nuc_sug_transp, PA, PAR1, PFK, PGI, PGK, PGM_PMM_1,PGM_PMM_II, PGM_PMM_III, PGM_PMM_IV, PP2C, PTR2, Peptidase_C26, Phi_1,Phytochrome, Pkinase, Pkinase_Tyr, Pollen_allerg_1, Pribosyltran,Proteasome, Pyr_redox, Pyr_redox_2, Pyr_redox_dim, RNA_pol_L,RNA_pol_Rpb6, RRM_1, RRN3, Radical_SAM, Ras, Response_reg, Rhodanese,Ribosomal_S8e, Rieske, SAC3_GANP, SBDS, SET, SRF-TF, SURF5, Skp1,Skp1_POZ, Ssl1, Sterol_desat, Sugar_tr, TCP, ThiF, Transaldolase,UQ_con, Ubie_methyltran, WD40, WRKY, adh_short, bZIP_1, bZIP_2,cNMP_binding, iPGM_N, p450, tRNA-synt_2b, ubiquitin, zf-A20, zf-AN1,zf-B_box, zf-C2H2, zf-C3HC4, zf-CCCH wherein the Pfam gathering cuttofffor said protein domain families is stated in Table 16; wherein saidplant cell is selected from a population of plant cells with saidrecombinant DNA by screening plants that are regenerated from plantcells in said population and that express said protein for an enhancedtrait as compared to control plants that do not have said recombinantDNA; and wherein said enhanced trait is selected from group of enhancedtraits consisting of enhanced water use efficiency, enhanced coldtolerance, enhanced heat tolerance, enhanced resistance to saltexposure, enhanced shade tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil.
 3. A plantcell with stably integrated, recombinant DNA that is to suppress thelevel of an endogenous protein having at least one domain of amino acidsin a sequence that exceeds that Pfam gathering cutoff for amino acidsequence alignment with a protein domain family identified by Pfam namein the group of Pfam names consisting of Catalase, Bromodomain, FTCD_N,MatE, DPBB_1, Pollen_allerg_1, tRNA-synt_2b, HGTP_anticodon, Sugar_tr,MFS_1, DUF6, DUF250, LEA_4, MIP, and DUF231 wherein the Pfam gatheringcutoff for said protein domain families is stated in Table 16; whereinsaid plant cells is selected from a population of plant cells with saidrecombinant DNA by screening plants that are regenerated from plantcells in said population and that the level of said protein issuppressed for an enhanced trait as compared to control plants that donot have said recombinant DNA; and wherein said enhanced trait isselected from the group of enhanced traits consisting of enhanced wateruse efficiency, enhanced cold tolerance, enhanced heat tolerance,enhanced resistance to salt exposure, enhanced shade tolerance,increased yield, enhanced nitrogen use efficiency, enhanced seed proteinand enhanced seed oil.